Methods for treating cancer

ABSTRACT

Disclosed herein are methods of treating one or more tumors by administering to the subject a therapeutically effective amount of a combination of RAD1901 or solvates (e.g., hydrate) or salts thereof and one or more second therapeutic agent(s) (e.g., everolimus). The cancer is an estrogen-dependent cancer, such as breast cancer, ovarian cancer, colon cancer, endometrial cancer, or prostate cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2016/030316, filed Apr. 29, 2016, which claims the benefit of U.S.Provisional Application No. 62/154,699, filed Apr. 29, 2015, U.S.Provisional Application No. 62/155,451, filed Apr. 30, 2015, U.S.Provisional Application No. 62/252,085, filed Nov. 6, 2015, U.S.Provisional Application No. 62/265,696, filed Dec. 10, 2015, U.S.Provisional Application No. 62/158,469, filed May 7, 2015, U.S.Provisional Application No. 62/252,916, filed Nov. 9, 2015, U.S.Provisional Application No. 62/265,774, filed Dec. 10, 2015, U.S.Provisional Application No. 62/192,940, filed Jul. 15, 2015, U.S.Provisional Application No. 62/265,658, filed Dec. 10, 2015, and U.S.Provisional Application No. 62/323,572, filed Apr. 15, 2016, U.S.Provisional Application No. 62/192,944, filed Jul. 15, 2015, U.S.Provisional Application No. 62/265,663, filed Dec. 10, 2015, and U.S.Provisional Application No. 62/323,576, filed Apr. 15, 2016, all ofwhich are incorporated herein by reference in their entireties.

BACKGROUND

Breast cancer is divided into three subtypes based on expression ofthree receptors: estrogen receptor (ER), progesterone receptor (PR), andhuman epidermal growth factor receptor-2 (Her2). Overexpression of ERsis found in many breast cancer patients. ER-positive (ER+) breastcancers comprise two-thirds of all breast cancers. Other than breastcancer, estrogen and ERs are associated with, for example, ovariancancer, colon cancer, prostate cancer and endometrial cancer.

ERs can be activated by estrogen and translocate into the nucleus tobind to DNA, thereby regulating the activity of various genes. See,e.g., Marino et al., “Estrogen Signaling Multiple Pathways to ImpactGene Transcription,” Curr. Genomics 7(8): 497-508 (2006); and Heldringet al., “Estrogen Receptors: How Do They Signal and What Are TheirTargets,” Physiol. Rev. 87(3): 905-931 (2007).

Agents that inhibit estrogen production, such as aromatase inhibitors(AIs, e.g., letrozole, anastrozole and aromasin), or those that directlyblock ER activity, such as selective estrogen receptor modulators(SERMs, e.g., tamoxifen, toremifene, droloxifene, idoxifene, raloxifene,lasofoxifene, arzoxifene, miproxifene, levormeloxifene, and EM-652 (SCH57068)) and selective estrogen receptor degraders (SERDs, e.g.,fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810),GW5638/DPC974, SRN-927, ICI182782 and AZD9496), have been usedpreviously or are being developed in the treatment of ER-positive breastcancers.

SERMs (e.g., tamoxifen) and AIs are often used as a first-line adjuvantsystemic therapy for ER-positive breast cancer. Tamoxifen is commonlyused for ER-positive breast cancer. AIs suppress estrogen production inperipheral tissues by blocking the activity of aromatase, which turnsandrogen into estrogen in the body. However, AIs cannot stop the ovariesfrom making estrogen, Thus, AIs are mainly used to treat postmenopausalwomen. Furthermore, as AIs are much more effective than tamoxifen withfewer serious side effects, AIs may also be used to treat premenopausalwomen with their ovarian function suppressed. See, e.g., Francis et al.,“Adjuvant Ovarian Suppression in Premenopausal Breast Cancer,” N. Engl.J. Med., 372:436-446 (2015).

While initial treatment with these agents may be successful, manypatients eventually relapse with drug-resistant breast cancers.Mutations affecting the ER have emerged as one potential mechanism forthe development of this resistance. See, e.g., Robinson et al.,“Activating ESR1 mutations in hormone-resistant metastatic breastcancer,” Nat. Genet. 45:1446-51 (2013). Mutations in the ligand-bindingdomain (LBD) of ER are found in 21% metastatic ER-positive breast tumorsamples from patients who received at least one line of endocrinetreatment. Jeselsohn, et al., “ESR1 mutations—a mechanism for acquiredendocrine resistance in breast cancer,” Nat. Rev. Clin. Oncol.,12:573-83 (2015).

Fulvestrant is currently the only SERD approved for the treatment ofER-positive metastatic breast cancers with disease progression followingantiestrogen therapy. Despite its clinical efficacy, the utility offulvestrant has been limited by the amount of drug that can beadministered in a single injection and by reduced bioavailability.Imaging studies using 18F-fluoroestradiol positron emission tomography(FES-PET) suggest that even at the 500 mg dose level, some patients maynot have complete ER inhibition, and insufficient dosing may be a reasonfor therapeutic failure.

Another challenge associated with estrogen-directed therapies is thatthey may have undesirable effects on uterine, bone, and other tissues.The ER directs transcription of estrogen-responsive genes in a widevariety of tissues and cell types. These effects can be particularlypronounced as endogenous levels of estrogen and other ovarian hormonesdiminish during menopause. For example, tamoxifen can cause bonethinning in premenopausal women and increase the risk of endometrialcancer because it acts as a partial agonist on the endometrium. Inpostmenopausal women, AIs can cause more bone loss and more broken bonesthan tamoxifen. Patients treated with fulvestrant may also be exposed tothe risk of osteoporosis due to its mechanism of action.

The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammaliantarget of rapamycin (mTOR) pathway is an intracellular signaling pathwayimportant in regulating the cell cycle. The frequent activation of thePI3K/AKT/mTOR pathway in cancer and its crucial role in cell growth andsurvival provide a challenge in finding an appropriate amount ofproliferation versus differentiation in order to utilize this balance inthe development of various therapies. See, e.g., Gitto et al., “Recentinsights into the pathophysiology of mTOR pathway dysregulation,” Res.Rep. Bio., 2:1-16 (2015).

Inhibitors of the PI3K pathway have shown the most promise when given incombination with other therapies. For example, everolimus, an allostericmTOR inhibitor, was approved in 2012 for use in combination with the AIexemestane for treating post-menopausal women with advanced hormonereceptor positive (HR+), HER2-breast cancer (BOLERO-2 study). Agentstargeting other components of the PI3K pathway are under development fortreating HR+ cancer, e.g., ATP-competitive dual inhibitors of PI3K andmTOR (e.g., BEZ235, GDC-0980), pan-PI3K inhibitors which inhibit allfour isoforms of class I PI3K (e.g., BKM120, GDC-0941), isoform-specificinhibitors of the various PI3K isoforms (e.g., BYL719, GDC-0032),allosteric and catalytic inhibitors of AKT (MK2206, GDC-0068,GSK2110183, GSK2141795, AZD5363), and ATP-competitive inhibitors of mTORonly (AZD2014, MLN0128, and CC-223). Dienstmann et al., “Picking thepoint of inhibition: a comparative review of PI3K/AKT/mTOR pathwayinhibitors,” Mol. Cancer Ther., 13(5):1021-31 (2014).

Despite their great potential, undesirable side effects associated withmTOR inhibitors have hindered their development as effective cancertherapies. Kaplan et al., “Strategies for the management of adverseevents associated with mTOR inhibitors,” Transplant Rev (Orlando),28(3): 126-133 (2014); and Pallet et al., “Adverse events associatedwith mTOR inhibitors,” Expert Opin. Drug Saf. 12(2): 177-186 (2013).

There remains a need for more durable and effective ER-targetedtherapies that can overcome challenges associated with the currentendocrine therapies, while providing additional benefits by combiningwith second therapeutic agents (e.g., everolimus and other agentstargeting the PI3K/AKT/mTOR pathway) to combat cancer in advanced stageand/or with resistance to prior treatments.

BRIEF SUMMARY OF THE INVENTION

One aspect of the invention relates to a method for treating one or morecancers and/or tumors in a subject comprising administering to thesubject a therapeutically effective amount of a combination of RAD1901or solvates (e.g., hydrate) or salts thereof and one or more secondtherapeutic agent(s) (e.g., everolimus) as described herein.

In some embodiments, the cancer is an estrogen-dependent cancer, such asbreast cancer, ovarian cancer, colon cancer, endometrial cancer, orprostate cancer. In some embodiments, the cancer is ER-positive breastcancer.

RAD1901 or solvates (e.g., hydrate) or salts thereof and the secondtherapeutic agent(s) (e.g., everolimus) are administered in combinationto a subject in need. The phrase “in combination” means that RAD1901 orsolvates (e.g., hydrate) or salts thereof may be administered before,during, or after the administration of the second therapeutic agent(s)(e.g., everolimus). For example, RAD1901 or solvates (e.g., hydrate) orsalts thereof and the second therapeutic agent(s) can be administeredabout one week apart, about 6 days apart, about 5 days apart, about 4days apart, about 3 days apart, about 2 days apart, about 24 hoursapart, about 23 hours apart, about 22 hours apart, about 21 hours apart,about 20 hours apart, about 19 hours apart, about 18 hours apart, about17 hours apart, about 16 hours apart, about 15 hours apart, about 14hours apart, about 13 hours apart, about 12 hours apart, about 11 hoursapart, about 10 hours apart, about 9 hours apart, about 8 hours apart,about 7 hours apart, about 6 hours apart, about 5 hours apart, about 4hours apart, about 3 hours apart, about 2 hours apart, about 1 hourapart, about 55 minutes apart, about 50 minutes apart, about 45 minutesapart, about 40 minutes apart, about 35 minutes apart, about 30 minutesapart, about 25 minutes apart, about 20 minutes apart, about 15 minutesapart, about 10 minutes apart, or about 5 minutes apart. In otherembodiments, RAD1901 or solvates (e.g., hydrate) or salts thereof andthe second therapeutic agent(s) are administered to the subjectsimultaneously or substantially simultaneously. In certain of theseembodiments, the compounds may be administered as part of a singleformulation.

In some embodiments, RAD1901 or solvates (e.g., hydrate) or saltsthereof and the second therapeutic agent(s) are administered in separateformulations. In certain of these embodiments, the formulations may beof the same type. For example, both formulations may be designed fororal administration (e.g., via two separate pills) or for injection(e.g., via two separate injectable formulations). In other embodiments,RAD1901 or solvates (e.g., hydrate) or salts thereof and the secondtherapeutic agent(s) may be formulated in different types offormulations. For example, one compound may be in a formulation designedfor oral administration, while the other is in a formulation designedfor injection.

In other embodiments, RAD1901 or solvates (e.g., hydrate) or saltsthereof and the second therapeutic agent(s) are administered as part ofa single formulation. For example, RAD1901 or solvates (e.g., hydrate)or salts thereof and the second therapeutic agent(s) are formulated in asingle pill for oral administration or in a single dose for injection.Accordingly, provided herein in certain embodiments are formulationscomprising RAD1901 or solvates (e.g., hydrate) or salts thereof and oneor more second therapeutic agents.

Administration routes of RAD1901 or solvates (e.g., hydrate) or saltsthereof and/or the second therapeutic agent(s) include but are notlimited to topical administration, oral administration, intradermaladministration, intramuscular administration, intraperitonealadministration, intravenous administration, intravesical infusion,subcutaneous administration, transdermal administration, andtransmucosal administration.

BRIEF DESCRIPTION OF DRAWINGS AND TABLES

This application contains at least one drawing executed in color. Copiesof this application with color drawing(s) will be provided by the Officeupon request and payment of the necessary fees.

FIG. 1: RAD1901 inhibited tumor growth in various patient-derivedxenograft (PDx) models regardless of ESR1 status and prior endocrinetherapy. Percentage of tumor growth inhibition (TGI) in PDx modelstreated with RAD1901 is shown.

FIGS. 2A-C: The combination of RAD1901 and everolimus demonstrated tumorgrowth inhibition and regression in wild-type (WT) ERα MCF-7 xenograftmodels (PR+, HER2−). (FIG. 2A): Tumor growth of MCF-7 xenograft modelstreated with vehicle control, everolimus (2.5 mg/kg, p.o., q.d),fulvestrant (3 mg/dose, s.c., qwk), a combination of fulvestrant (3mg/dose, s.c., qwk) and everolimus (2.5 mg/kg, p.o., q.d), RAD1901 (60mg/kg, p.o., q.d.), and a combination of RAD1901 (60 mg/kg, p.o., q.d.)and everolimus (2.5 mg/kg, p.o., q.d); One-way ANOVA, “ns” is notsignificant, *p-value<0.05, and ***p-value<0.001; (FIG. 2B): Change inindividual tumor size from baseline to end of study of MCF-7 xenograftmodels treated with vehicle control, everolimus (2.5 mg/kg, p.o., q.d),fulvestrant (3 mg/dose, s.c., qwk), a combination of fulvestrant (3mg/dose, s.c., qwk) and everolimus (2.5 mg/kg, p.o., q.d), RAD1901 (60mg/kg, p.o., q.d.), and combinations of RAD1901 (60 mg/kg, p.o., q.d.)and everolimus (2.5 mg/kg, p.o., q.d); (FIG. 2C): Tumor growth of MCF-7xenograft models treated with vehicle control, everolimus (2.5 mg/kg,p.o., q.d), fulvestrant (3 mg/dose, s.c., qwk), a combination offulvestrant (3 mg/dose, s.c., qwk) and everolimus (2.5 mg/kg, p.o.,q.d), RAD1901 (30 or 60 mg/kg, p.o., q.d.), and a combination of RAD1901(30 or 60 mg/kg, p.o., q.d.) and everolimus (2.5 mg/kg, p.o., q.d).

FIGS. 3A-B: The combination of RAD1901 and everolimus demonstrated tumorgrowth inhibition and regression in WT ERα PDx-11 models (PR+, Her2+,previously treated with aromatase inhibitor, fulvestrant, andchemotherapy). (FIG. 3A): Tumor growth of PDx-11 models treated withvehicle control, fulvestrant (3 mg/dose, s.c., qwk), everolimus (2.5mg/kg, p.o., q.d), RAD1901 (60 mg/kg, p.o., q.d.), and a combination ofRAD1901 (60 mg/kg, p.o., q.d.) and everolimus (2.5 mg/kg, p.o., q.d);(FIG. 3B): Change in individual tumor size from baseline to end of studyin PDx-11 models treated with vehicle control, fulvestrant (3 mg/dose,s.c., qwk), RAD1901 (60 mg/kg, p.o., q.d.), and a combination of RAD1901(60 mg/kg, p.o., q.d.) and everolimus (2.5 mg/kg, p.o., q.d).n=8-10/group.

FIGS. 4A-B: The combination of RAD1901 and everolimus demonstrated tumorgrowth inhibition in WT ER+PDx-2 models (PR+, Her2+, treatment naïve).(FIG. 4A): Tumor growth of PDx-2 models treated with vehicle control,RAD1901 (60 mg/kg, p.o., q.d.), fulvestrant (3 mg/dose, s.c., qwk), anda combination of RAD1901 (60 mg/kg, p.o., q.d.) and fulvestrant (3mg/dose, s.c., qwk); (FIG. 4B): Tumor growth of PDx-2 models treatedwith vehicle control, everolimus (2.5 mg/kg, p.o., q.d), RAD1901 (60mg/kg, p.o., q.d.), and a combination of RAD1901 (60 mg/kg, p.o., q.d.)and everolimus (2.5 mg/kg, p.o., q.d). n=8-10/group.

FIG. 5: Efficacy of RAD1901 sustained at least two months after RAD1901treatment ended while estradiol treatment continued in WT ERα PDx-4models (PR+, Her2+, treatment naïve).

FIGS. 6A-B: The combination of RAD1901 and everolimus demonstrated tumorgrowth inhibition in mutant (Y537S) ERα PDx-5 models (PR+, Her2+,previously treated with aromatase inhibitors). (FIG. 6A): Tumor growthof PDx-5 models treated with vehicle control, RAD1901 (60 mg/kg, p.o.,q.d.), and fulvestrant (3 mg/kg, s.c., qwk); (FIG. 6B): Tumor growth ofPDx-5 models treated with vehicle control, RAD1901 (60 mg/kg, p.o.,q.d.), everolimus (2.5 mg/kg, p.o., q.d), and a combination of RAD1901(60 mg/kg, p.o., q.d.) and everolimus (2.5 mg/kg, p.o., q.d).n=8-10/group.

FIG. 7: Pharmacokinetic analysis of fulvestrant in nude mice. The plasmaconcentration of fulvestrant at 1 mg/dose (solid diamond), 3 mg/dose(solid circle), and 5 mg/dose (solid triangle) is shown. The nude micewere dosed subcutaneously with fulvestrant on Day 1 and the second doseon Day 8. The plasma concentration of fulvestrant was monitored at theindicated time points for up to 168 hours after the second dose.

FIG. 8: Effect of RAD1901 and fulvestrant (Faslodex) on mouse survivalin an intracranial MCF-7 tumor model.

FIGS. 9A-C: A representative image of FES-PET scan of the uterus of asubject treated with 200 and 500 mg RAD1901 p.o., q.d., and change ofthe ER engagement after the RAD1901 treatments. (FIG. 9A): Transversalview of uterus CT scan before 200 mg RAD1901 treatment (a) and after(c), and transversal view of uterus FES-PET scan before the RAD1901treatment (b) and after (d); (FIG. 9B): Sagittal view of uterus CT scanbefore 500 mg RAD1901 treatment (top (a) panel) and after (bottom (a)panel), sagittal view of uterus FES-PET scan before the RAD1901treatment (top (b) panel) and after (bottom (b) panel), transversal viewof uterus CT scan before the RAD1901 treatment (top (c) panel) and after(bottom (c) panel), transversal view of uterus FES-PET scan before theRAD1901 treatment (top (d) panel) and after (bottom (d) panel); (FIG.9C): % change of ER engagement after the RAD1901 treatments of Subjects1-3 (200 mg) and Subjects 4-7 (500 mg) compared to baseline (beforeRAD1901 treatment).

FIGS. 10A-B: A representative image of FES-PET scan of the uterus (FIG.10A) and pituitary (FIG. 10B) before (Baseline) and after(Post-treatment) RAD1901 treatment (500 mg). (a) Lateral cross-section;(b) longitude cross-section; and (c) longitude cross-section.

FIG. 11: PR and ER expression in MCF-7 xenograft models treated withvehicle control, RAD1901, everolimus, a combination of RAD1901 andeverolimus, fulvestrant, and a combination of fulvestrant andeverolimus.

FIGS. 12A-B: RAD1901 treatment resulted in complete ER degradation andinhibited ER signaling in MCF-7 cell lines (FIG. 12A) and T47D celllines (FIG. 12B) in vitro. The ER expression was shown in both celllines treated with RAD1901 and fulvestrant at various concentrations of0.001 μM, 0.01 μM, 0.1 μM and 1 μM, respectively. ER signaling was shownby three ER target genes tested: PGR, GREB1 and TFF1.

FIGS. 13A-C: RAD1901 treatment resulted in ER degradation and abrogationof ER signaling in MCF-7 xenograft models. (FIG. 13A): Western blotshowing PR and ER expression in the MCF-7 xenograft models treated withvehicle control, RAD1901 at 30 and 60 mg/kg, and fulvestrant at3mg/dose, 2 hour or 8 hour after the last dose; (FIG. 13B): ER proteinexpression in the MCF-7 xenograft models treated with vehicle control,RAD1901 at 30 and 60 mg/kg, and fulvestrant at 3mg/dose, 2 hour afterthe last dose; (FIG. 13C): PR protein expression in the MCF-7 xenograftmodels treated with vehicle control, RAD1901 at 30 and 60 mg/kg, andfulvestrant at 3mg/dose, 8 hour after last dose.

FIGS. 14A-C: RAD1901 treatment resulted in a rapid decrease in PR inMCF-7 xenograft models. (FIG. 14A): Western blot showing PR expressionin MCF-7 xenograft models treated with vehicle control and RAD1901 at30, 60, and 90 mg/kg, at 8 hours or 12 hours after single dose; (FIG.14B): Western blot showing PR expression in MCF-7 xenograft modelstreated with vehicle control and RAD1901 at 30, 60, and 90 mg/kg, at 4hours or 24 hours after the 7th dose; (FIG. 14C): Dose-dependentdecrease in PR expression in MCF-7 xenograft models treated with RAD1901at 30, 60, and 90 mg/kg.

FIGS. 15A-B: RAD1901 treatment resulted in a rapid decrease inproliferation in MCF-7 xenograft models. (FIG. 15A): A representativephotograph of a sectioned tumor harvested from MCF-7 xenograft modelstreated with vehicle control and RAD1901 at 90 mg/kg, 8 hours aftersingle dose and 24 hours after the 4th dose, stained for proliferationmarker Ki-67; (FIG. 15B): Histogram showing decrease of proliferationmarker Ki-67 in MCF-7 xenograft models treated with vehicle control andRAD1901 at 90 mg/kg, 8 hours after single dose and 24 hours after the4th dose.

FIG. 16: RAD1901 treatment at 30, 60, and 120 mg/kg decreased Ki67 moresignificantly than fulvestrant (1 mg/animal) in end of study tumors ofPDx-4 models four hours on the last day of a 56 day efficacy study.

FIG. 17: RAD1901 treatment at 60 and 120 mg/kg resulted in reduced ERsignaling in vivo in PDx-5 models with decreased PR expression.

FIGS. 18A-D: Effect of RAD1901 on uterine tissue in newly weaned femaleSprague-Dawley rats. (FIG. 18A): Uterine wet weights of rats euthanized24 hours after the final dose; (FIG. 18B): Epithelial height in tissuesections of the uterus; (FIG. 18C): Representative sections of ToluidineBlue O-stained uterine tissue at 400× magnification, arrows indicateuterine epithelium; (FIG. 18D): Total RNA extracted from uterine tissueand analyzed by quantitative RT-PCR for the level of complement C3expression relative to the 18S ribosomal RNA housekeeping gene.

FIG. 19: Plasma pharmacokinetic results of RAD1901 at 200, 500, 750, and1000 mg/kg after dosing on Day 7.

FIG. 20: 3ERT (I).

FIG. 21: 3ERT (II).

FIG. 22: Superimpositions of the ERα LBD-antagonist complexes summarizedin Table 11.

FIGS. 23A-B: Modeling of (FIG. 23A) RAD1901-1R5K; and (FIG. 23B)GW5-1R5K.

FIGS. 24A-B: Modeling of (FIG. 24A) RAD1901-1SJ0; and (FIG. 24B)E4D-1SJ0.

FIGS. 25A-B: Modeling of (FIG. 25A) RAD1901-2JFA; and (FIG. 25B)RAL-2JFA.

FIGS. 26A-B: Modeling of (FIG. 26A) RAD1901-2BJ4; and (FIG. 26B)OHT-2BJ4.

FIGS. 27A-B: Modeling of (FIG. 27A) RAD1901-2IOK; and (FIG. 27B)IOK-2IOK.

FIG. 28: Superimpositions of the RAD1901 conformations resulted from IFDanalysis with 1R5K and 2OUZ.

FIG. 29: Superimpositions of the RAD1901 conformations resulted from IFDanalysis with 2BJ4, and 2JFA.

FIGS. 30A-B: Superimpositions of the RAD1901 conformations resulted fromIFD analysis with 2BJ4, 2JFA and 1SJ0.

FIGS. 31A-C: IFD of RAD1901 with 2BJ4.

FIGS. 32A-C: Protein Surface Interactions of RAD1901 docked in 2BJ4 byIFD.

FIGS. 33A-C: IFD of Fulvestrant with 2BJ4.

FIGS. 34A-B: IFD of Fulvestrant and RAD1901 with 2BJ4.

FIGS. 35A-B: Superimposions of IFD of Fulvestrant and RAD1901 with 2BJ4.

FIG. 36: RAD1901 in vitro binding assay with ERα constructs of WT andLBD mutant.

Table 1. RAD1901 levels in plasma, tumor and brain of mice implantedwith MCF7 cells after treated for 40 days. BLQ: below the limit ofquantitation.

Table 2. SUV for uterus, muscle, and bone for a human subject treatedwith 200 mg dose PO once/day for six days.

Table 3. SUV for uterus, muscle, and bone for a human subjects (n=4)treated with 500 mg dose PO once/day for six days.

Table 4. Effect of RAD1901 on BMD in ovariectomized rats. Adult femalerats underwent either sham or ovariectomy surgery before treatmentinitiation with vehicle, E2 (0.01 mg/kg) or RAD1901 (3 mg/kg) once daily(n=20 per treatment group). BMD was measured by dual emission x-rayabsorptiometry at baseline and after 4 weeks of treatment. Data areexpressed as mean±SD. *P<0.05 versus the corresponding OVX+Veh control.BMD, bone mineral density; E2, beta estradiol; OVX, ovariectomized; Veh,vehicle.

Table 5. Effect of RAD1901 on femur microarchitecture in ovariectomizedrats. Adult female rats underwent either sham or ovariectomy surgerybefore treatment initiation with vehicle, E2 (0.01 mg/kg) or RAD1901 (3mg/kg) once daily (n=20 per treatment group). After 4 weeks, Bonemicroarchitecture was evaluated using microcomputed tomography. Data areexpressed as mean±SD. *P<0.05 versus the corresponding OVX+Veh control.ABD, apparent bone density; BV/TV, bone volume density; ConnD,connectivity density; E2, beta estradiol; OVX, ovariectomized; TbN,trabecular number; TbTh, trabecular thickness; TbSp, trabecular spacing;Veh, vehicle.

Table 6. Key baseline demographics of Phase 1 dose escalation study ofRAD1901.

Table 7. Most frequent (>10%) treatment related AEs in a Phase 1 doseescalation study of RAD1901. AEs graded as per CTCAE v4.0. Any patientwith multiple scenarios of a same preferred term was counted only onceto the most severe grade. *>10% of patients in the total active groupwho had any related TEAEs. N=number of subjects with at least onetreatment-related AE in a given category.

Table 8. Pharmacokinetic parameters in a Phase 1 dose escalation studyof RAD1901 (Day 7).

Table 9. Frequency of LBD mutations.

Table 10. Differences of ER-α LBD-antagonist complexes in residue posesversus 3ERT.

Table 11. Evaluation of structure overlap of ER-α LBD-antagonistcomplexes by RMSD calculations.

Table 12. Analysis of ligand binding in ER-α LBD-antagonist complexes.

Table 13. Model evaluation for RAD1901 docking.

Table 14. Induced Fit Docking Score of RAD1901 with 1R5K, 1SJ0, 2IFA,2BJ4 and 2OUZ.

DETAILED DESCRIPTION OF THE INVENTION

As set forth in the Examples section below, a combination of RAD1901 andeverolimus (a RAD1901-everolimus combination) (structures below)demonstrated greater tumor growth inhibition than RAD1901 alone inseveral breast cancer xenograft models, including a wild-type (WT) ERαMCF-7 xenograft model (FIGS. 2A-C), WT ERα PDx-2 (FIGS. 4A-B) and PDx-11models (FIGS. 3A-B), and a mutant (e.g., Y537S) ERα PDx-5 model (FIGS.6A-B), regardless of ESR1 status, and prior endocrine therapy asdescribed in Example I. PDx-2, PDx-5 and PDx-11 models had tumorexpressing WT or mutant (e.g., Y537S) ERα, with PR expression, with highor low Her2 expression, and with or without prior endocrine therapy(e.g., AI, fulvestrant), and/or chemotherapy (chemo) (FIG. 1). RAD1901alone also inhibited tumor growth in all other PDx models listed in FIG.1, having tumor expressing WT or mutant (e.g., Y537S) ERα, with PRexpression, with high or low Her2 expression, and with or without priorendocrine therapy (e.g., tamoxifen (tam), AI, fulvestrant), chemotherapy(chemo), Her2 inhibitors (Her2i, e.g., trastuzumab, lapatinib),bevacizumab, and/or rituximab.

ER WT PDx models and ER mutant PDx models may have different level ofresponsiveness to treatment with fulvestrant alone, everolimus alone,and/or a combination of fulvestrant and everolimus (a ful-everolimuscombination). However, RAD1901-everolimus combinations demonstratedimproved tumor growth inhibition and/or tumor regression compared totreatment with RAD1901 alone or everolimus alone, regardless of whetherthe PDx models were responsive to fulvestrant treatment and/orful-everolimus combination treatment. In other words, RAD1901-everolimuscombination may inhibit tumor growth and/or produce tumor regression infulvestrant resistant cancers.

RAD1901-everolimus combination treatment demonstrated improved tumorgrowth inhibition and/or tumor regression compared to treatment withfulvestrant alone or with the ful-everolimus combination. For example,the RAD1901-everolimus combination caused more significant tumorregression in more WT ER+xenograft models than treatment withfulvestrant alone, RAD1901 alone, or everolimus alone, even though thesexenograft models have varied responsiveness to fulvestrant treatment(e.g., MCF-7 cell line xenograft model responsive to fulvestranttreatment (FIGS. 2A-C); PDx-11 model responsive to fulvestrant treatment(FIGS. 3A-B); and PDx-2 model least responsive to fulvestrant treatment(FIGS. 4A-B). The RAD1901-everolimus combination also caused moresignificant tumor regression in more WT ER+MCF-7 cell line xenograftmodels and PDx-11 models than treatment with a ful-everolimuscombination (FIGS. 2A-C and 3A-B). The RAD1901-everolimus combinationprovided similar effects with RAD1901 at a dose of 30 mg/kg or 60 mg/kg,although RAD1901 alone at 30 mg/kg was not as effective as RAD1901 aloneat 60 mg/kg in inhibiting tumor growth (FIG. 2C). Said results suggest aRAD1901-everolimus combination with a lower dose of RAD1901 (e.g., 30mg/kg) was sufficient to maximize the tumor growth inhibition/tumorregression effects in said xenograft models.

The RAD1901-everolimus combination demonstrated tumor regression orimproved tumor growth inhibition in mutant ER+(e.g., Y537S) PDx modelshardly responsive to fulvestrant treatment (FIG. 6A). For example, PDx-5is an ER Y537S mutant PDx model (PR+, Her2-, prior treatment with AI)hardly responsive to fulvestrant treatment. RAD1901-everolimuscombination demonstrated tumor regression in PDx-5 model, whileeverolimus alone or RAD1901 alone only inhibited tumor growth withoutcausing tumor regression (FIG. 6B). The RAD1901-everolimus combinationcaused more significant tumor growth inhibition than RAD1901 alone,everolimus alone, or fulvestrant alone in mutant PDx-5 models (FIG. 6B).Thus, the addition of everolimus benefited the PDx-5 models when appliedin combination with RAD1901. Thus, RAD1901-everolimus combinationsprovide powerful anti-tumor therapy for ER+breast cancer expressing WTor mutant ER, with PR expression, with high or low Her2 expression, andwith or without resistance to fulvestrant.

The results provided herein also show that RAD1901 can be delivered tothe brain (Example II), and that said delivery improved mouse survivalin an intracranial tumor model expressing wild-type ERα (MCF-7 xenograftmodel, Example I(B)). Everolimus was approved to treat subependymalgiant cell astrocytoma (SEGA), a brain tumor seen with tuberoussclerosis (TS). Thus, both components of a RAD1901-everolimuscombination are likely to be able to cross the brain-blood barrier andtreat ER+ tumors in brain. This represents an additional advantage overthe ful-everolimus combination for treating ER+ tumors in the brain, asfulvestrant cannot cross the blood-brain barrier (Vergotel et al.,“Fulvestrant, a new treatment option for advanced breast cancer:tolerability versus existing agents,” Ann. Oncol., 17(2):200-204(2006)). A combination of RAD1901 with other second therapeutic agent(s)that can cross the blood-brain barrier (e.g., mTOR inhibitors such asrapamycin analogs (Geoerger et al., “Antitumor activity of the rapamycinanalog CCI-779 in human primitive neuroectodermal tumor/medulloblastomamodels as single agent and in combination chemotherapy,” Cancer Res.61:1527-1532 (2001))) may also have similar therapeutic effects on ER+tumors in brain.

RAD1901 showed sustained efficacy in inhibiting tumor growth afterRAD1901 treatment ended while estradiol treatment continued (e.g., PDx-4model). Thus, a RAD1901-everolimus combination is likely to benefitpatients by inhibiting tumor growth after treatment ends, especiallywhen the second therapeutic agent(s) treatment may be discontinued(e.g., 29% for everolimus) or reduced or delayed (70% foreverolimus-treated patients) for adverse reactions.http://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm488028.htm.

A RAD1901-everolimus combination is likely to have fewer and/or lesssevere side-effects than treatment with everolimus alone or acombination of everolimus with other hormone therapies (e.g., AIs suchas letrozole and SERDs such as fulvestrant). For example, both AIs andfulvestrant may cause bone loss in treated patients. RAD1901 is unlikelyto have similar side effects. RAD1901 was found to preferentiallyaccumulate in tumor, with a RAD1901 level in tumor v. RAD1901 level inplasma (T/P ratio) of up to about 35 (Example II). Standardized uptakevalues (SUV) for uterus, muscle and bone were calculated for humansubjects treated with RAD1901 at a daily dose of about 200 mg up toabout 500 mg (Example III(A)). Post-dose uterine signals were close tolevels from “non-target tissues” (tissues that do not express estrogenreceptor), suggesting a complete attenuation of FES-PET uptakepost-RAD1901 treatment. Almost no change was observed in pre-versuspost-treatment PET scans in tissues that did not significantly expressestrogen receptor (e.g., muscles, bones) (Example IIIA). Finally,RAD1901 treatments antagonized estradiol stimulation of uterine tissuesin ovariectomized (OVX) rats (Example IV(A)), and largely preserved bonequality of the treated subjects. For example, OVX rats treated withRAD1901 showed maintained BMD and femur microarchitecture (ExampleIV(A)). Thus, the RAD1901-everolimus combination may be especiallyuseful for patients having osteoporosis or a higher risk ofosteoporosis.

Furthermore, gene expression profiling has been reported as effectivefor identifying patients responsive to everolimus treatment. Yoon etal., “Gene expression profiling identifies responsive patients withcancer of unknown primary treated with carboplatin, paclitaxel, andeverolimus: NCCTG NO871 (alliance),” Ann. Oncol., 27(2):339-44 (2016).Study NCT00805129 found everolimus is more efficient in patients thatpresent somatic mutations in TSC1 as said mutations lead to an increasein recurrence and to an increase in the response time to everolimus.Thus, methods disclosed herein may further comprise gene profiling ofsubjects to be treated in order to identify subjects with greaterresponse and/or longer responsive time.

Furthermore, RAD1901 was found to degrade wild-type ERα and abrogate ERsignaling in vivo in MCF-7 cell line xenograft models, and produced adose-dependent decrease in PR in these MCF-7 cell line xenograft models(Example 111(B)). RAD1901 decreased proliferation in MCF-7 cell linexenograft models and PDx-4 models as evidenced by a decrease inproliferation marker Ki67 in tumors harvested from the treated subjects.RAD1901 also decreased ER signaling in vivo in an ER mutant PDx modelthat was hardly responsive to fulvestrant treatment (Example 111(B)).

The unexpected efficacy of the RAD1901-everolimus combination intreating tumors hardly responsive to fulvestrant treatments and intumors expressing mutant ERα may be due to the unique interactionsbetween RAD1901 and ERα. Structural models of ERα bound to RAD1901 andother ERα-binding compounds were analyzed to obtain information aboutthe specific binding interactions (Example V). Computer modeling showedthat RAD1901-ERα interactions are not likely to be affected by mutationsin the LBD of ERα, e.g., Y537X mutant wherein X was S, N, or C; D538G;and S463P, which account for about 81.7% of LBD mutations found in arecent study of metastatic ER positive breast tumor samples frompatients who received at least one line of endocrine treatment (Table 9,Example V). Thus, a combination of one or more second therapeuticagent(s) (e.g., everolimus) and RAD1901 or salt or solvate (e.g.,hydrate) thereof is likely to have therapeutic effects with relativelylow side effects similar to RAD1901-everolimus combinations as disclosedherein. The computer modeling resulted in identification of specificresidues in the C-terminal ligand-binding domains of ERα that arecritical to binding, information that can be used to develop compoundsthat bind and antagonize not only wild-type ERα but also certain mutantsand variants thereof, which when combined with a second therapeuticagent (e.g., everolimus) may provide strong anti-tumor therapy withrelatively low side effects similar to RAD1901-everolimus combinationsas disclosed herein.

Based on the results provided herein, methods are provided forinhibiting growth or producing regression of an ERα-positive tumor in asubject in need thereof by administering to the subject atherapeutically effective amount of a combination of RAD1901 or solvates(e.g., hydrates) or salts thereof, plus one or more second therapeuticagent(s) as described herein (e.g., everolimus).

In certain embodiments, administration of RAD1901 or salt or solvate(e.g., hydrate) thereof has additional therapeutic benefits in additionto inhibiting tumor growth, including for example inhibiting cancer cellproliferation or inhibiting ERα activity (e.g., by inhibiting estradiolbinding or by degrading ERα). In certain embodiments, the methodproduces little or no negative effects on non-targeted tissues (e.g.,muscles, bones).

In certain embodiments, RAD1901 or salt or solvate (e.g., hydrate)thereof modulates and/or degrades ERα and mutant ERα.

In certain embodiments of the tumor growth inhibition or tumorregression methods provided herein, methods are provided for inhibitinggrowth or producing regression of an ERα-positive tumor in a subject inneed thereof by administering to the subject a therapeutically effectiveamount of a combination of RAD1901 or a solvate (e.g., hydrate) or saltthereof and one or more second therapeutic agent(s) as described herein.In certain of these embodiments, the salt thereof is RAD1901dihydrochloride having the structure:

Second Therapeutic Agents

A second therapeutic agent for use in the methods provided herein can bea chemotherapeutic agent, or an inhibitor of AKT, androgen receptor,angiogenesis, aromatase, aurora A kinase, BCL2, EGFR, the estrogenpathway, estrogen signaling pathway, estrogen receptor, HER2, HER3, heatshock protein 90 (Hsp90), hedgehog (Hh) signaling pathway, histonedeacetylase (HDAC), KIT pathways, mTOR (e.g., TORC1 and/or TORC2),microtubule, MYC, nucleoside metabolism, PARP, pan PI3K, PI3K, proteinkinase CK2, the RAS pathway, steroid sulfatase (STS), TK, Top2A,tyrosine kinase, VEGF receptor tyrosine kinase, or any combinationsthereof. The second therapeutic agent may also be an antibody such as ananti-TGF beta antibody, anti-type-1 insulin like growth factor receptorantibody, anti-TROP-2 antigen antibody, anti-HER3 antibody, anti-PD1antibody, or a drug conjugate thereof.

Further examples of second therapeutic agents include, withoutlimitation, abiraterone acetate, ADI-PEG 20, ado-trastuzumab emtansine,afatinib, alisertib, anastrozole, paclitaxel, and paclitaxel derivatives(e.g., ANG1005, paclitaxel polymeric micelle), ARN-810, azacitidine,AZD2014, AZD5363, bevacizumab, BP-C1, buparlisib (BKM120), BYL719,capecitabine, carboplatin, cediranib Maleate, cetuximab,cisplatin/AC4-CDDP4, CR1447, CX-4945, dasatinib, denosumab, docetaxel,doxorubicin, eniluracil, entinostat, enzalutamide, epirubicin, eribulin,exemestane, everolimus, flourouracil, fulvestrant, fresolimumab,ganetespib, ganitumab, GDC-0032, GDC-0941, gemcitabine, glembatumumabvedotin, GnRH agonist (e.g. goserelin acetate), GRN1005, GSK 2141795,ibandronate, IMMU-132, irinotecan, irosustat, epothilone (e.g.,ixabepilone), lapatinib, sonidegib (LDE225), letrozole, LGK974, LJM716,lucitanib, methotrexate, MK-2206, MK-3475, MLN0128, MM-302, neratinib,niraparib, olaparib, anti-androgen (e.g., orteronel), oxaliplatin,pazopanib, pertuzumab, PF-05280014, PM01183, progesterone, pyrotinib,romidepsin, ruxolitinib, sorafenib, sunitinib, talazoparib, tamoxifen,taxane, T-DM1, telapristone (CDB-4124), temozolomide, temsirolimus,terathiomolybdate, tesetaxel, TLR 7 agonist, TPI 287, trametinib,trastuzumab, TRC105, trebananib (AMG 386), triptorelin, veliparib,vinflunine, vinorelbine, vorinostat, zoladex, and zoledronic acid,including solvates (e.g., hydrates) and salts thereof.

In certain embodiments, the second therapeutic agents are selected fromthe group consisting of ado-trastuzumab emtansine, aurora A kinaseinhibitors (e.g., alisertib), AIs (e.g., anastrozole; exemestane,letrozole), ARN-810, mTOR inhibitors (e.g., everolimus, AZD2014, BEZ235,GDC-0980, CC-223, MLN0128), AKT inhibitors (e.g., AZD5363, GDC-0068,GSK2110183, GSK2141795, GSK690693, MK2206), PI3K inhibitors (e.g.,BKM120, BYL719, GDC-0032, GDC-0941), selective histone deacetylase(HDAC) inhibitors (e.g., entinostat), GnRH agonist (e.g., goserelinacetate), GRN1005 and combinations thereof with trastuzumab, lapatinib,tyrosine kinase inhibitor (e.g., lucitanib, neratinib), anti-androgen(e.g., orteronel), pertuzumab, temozolomide, and antibodies (e.g.,keytruda and BYM338).

In certain embodiments, the second therapeutic agent can be an AI (e.g.,anastrozole, aromasin, and letrozole), another SERM (e.g., arzoxifene,droloxifene, EM-652 (SCH 57068), idoxifene, lasofoxifene,levormeloxifene, miproxifene, raloxifene, tamoxifen, and toremifene), oranother SERD (e.g., fulvestrant, GDC-0810 (ARN-810), GW5638/DPC974,ICI182782, RU58668, SRN-927, TAS-108 (SR16234), and ZK191703), includingsolvates (e.g., hydrates) and salts thereof.*

Further examples of the second therapeutic agents include, withoutlimitation, abraxane, AMG 386, cabazitaxel, caelyx, capecitabine,docetaxel, eribulin, gemcitabine, herceptin, neratinib, pazopanib(GW786034), rapalogs (rapamycin and its analogs), taxol (includinganalogs/alternative formulations), TDM1, temozolamide, tykerb, veliparib(ABT-888), and vinorelbine, including solvates (e.g., hydrates) andsalts thereof.

Second Therapeutic Agent Targeting the PI3K/AKT/mTOR Pathway

In some embodiments, the second therapeutic agent targets thePI3K/AKT/mTOR pathway and can be a mTOR inhibitor, a dual mTORinhibitor, a PI3K/mTOR inhibitor. In some embodiments, the secondtherapeutic agent is a rapamycin derivative (aka rapalog) such asrapamycin (sirolimus or rapamune, Pfizer), everolimus (Afinitor orRAD001, Novartis), ridaforolimus (AP23573 or MK-8669, Merck and ARIADPharmaceuticals), temsirolimus (Torisel or CCI779, Pfizer), includingsolvates (e.g., hydrates) and salts thereof. In some embodiments, thesecond therapeutic agent is a dual mTOR inhibitor that inhibits bothmTORC1 and mTORC2, such as MLN0128 (castration-resistant prostate cancer(CRPC), Memorial Sloan Kettering Cancer Center), CC115 and CC223(Celgene), OSI-027 (OSI Pharmaceuticals), and AZD8055 and AZD2014(AstraZeneca), including solvates (e.g., hydrates) and salts thereof. Insome embodiments, the second therapeutic agent is a PI3K/mTOR inhibitorsuch as GDC-0980, SAR245409 (XL765), LY3023414 (Eli Lilly), NVP-BEZ235(Novartis), NVP-BGT226 (Novartis), SF1126, and PKI-587 (Pfizer),including solvates (e.g., hydrates) and salts thereof

In certain embodiments, more than one of the second therapeutic agentsdisclosed above may be used in combination with RAD1901 or solvates(e.g., hydrate) or salts thereof. For example, an mTOR inhibitor can beused together with another mTOR inhibitor or with a PI3K/mTOR inhibitor.Also, it is known in the art that the second therapeutic agentsdisclosed above, including mTOR inhibitors, dual mTOR inhibitors, andPI3K/mTOR inhibitors, can be administered with other active agents toenhance the efficacy of the treatment. For example, an mTOR inhibitorcan be used in combination with JAK2 inhibitors (Bogani et al., PLOSOne, 8(1): e54826 (2013)), chemotherapeutic agents (Yardley, BreastCancer (Auckl) 7: 7-22 (2013)), or endocrine therapies such as tamoxifenor exemestane (Vinayak et al., “mTOR inhibitors in the treatment ofbreast cancer,” Oncology, published Jan. 15, 2013(http://www.cancernetwork.com/breast-cancer/mtor-inhibitors-treatment-breast-cancer)).Accordingly, the second therapeutic agents also include these auxiliaryactive agents.

Combination Therapy

(1) Combination of RAD1901 or solvates (e.g., hydrate) or salts thereofand One or More second Therapeutic Agent(s)

Both the RAD1901 or solvates (e.g., hydrate) or salts thereof and thesecond therapeutic agent(s) (e.g., everolimus), when administered aloneto a subject, have a therapeutic effect on one or more cancers or tumors(Examples I(A) and I(B)). It was surprisingly discovered that whenadministered in combination to a subject, RAD1901 or solvates (e.g.,hydrate) or salts thereof and the second therapeutic agent(s) (e.g.,everolimus) have a significantly improved effect on the cancers/tumors(Examples I(A) and I(B)).

“Inhibiting growth” of an ERα-positive tumor as used herein may refer toslowing the rate of tumor growth, or halting tumor growth entirely.

“Tumor regression” or “regression” of an ERα-positive tumor as usedherein may refer to reducing the maximum size of a tumor. In certainembodiments, administration of a combination of one or more secondtherapeutic agent(s) (e.g., everolimus) as described herein (e.g.,ribociclib, abemaciclib and everolimus) and RAD1901 or a solvate (e.g.,hydrate) or salt thereof may result in a decrease in tumor size versusbaseline (i.e., size prior to initiation of treatment), or eveneradication or partial eradication of a tumor. Accordingly, in certainembodiments the methods of tumor regression provided herein may bealternatively characterized as methods of reducing tumor size versusbaseline.

“Tumor” as used herein is a malignant tumor, and is used interchangeablywith “cancer.”

Tumor growth inhibition or regression may be localized to a single tumoror to a set of tumors within a specific tissue or organ, or may besystemic (i.e., affecting tumors in all tissues or organs).

As RAD1901 is known to preferentially bind ERα versus estrogen receptorbeta (ERβ), unless specified otherwise, estrogen receptor, estrogenreceptor alpha, ERα, ER, wild-type ERα, and ESR1 are usedinterchangeably herein. “Estrogen receptor alpha” or “ERα” as usedherein refers to a polypeptide comprising, consisting of, or consistingessentially of the wild-type ERα amino acid sequence, which is encodedby the gene ESR1. A tumor that is “positive for estrogen receptoralpha,” “ERα-positive,” “ER+,” or “ERα+” as used herein refers to atumor in which one or more cells express at least one isoform of ERα. Incertain embodiments, these cells overexpress ERα. In certainembodiments, the patient has one or more cells within the tumorexpressing one or more forms of ERβ. In certain embodiments, theERα-positive tumor and/or cancer is associated with breast, uterine,ovarian, or pituitary cancer. In certain of these embodiments, thepatient has a tumor located in breast, uterine, ovarian, or pituitarytissue. In those embodiments where the patient has a tumor located inthe breast, the tumor may be associated with luminal breast cancer thatmay or may not be positive for HER2, and for HER2+ tumors, the tumorsmay express high or low HER2 (e.g., FIG. 1). In other embodiments, thepatient has a tumor located in another tissue or organ (e.g., bone,muscle, brain), but is nonetheless associated with breast, uterine,ovarian, or pituitary cancer (e.g., tumors derived from migration ormetastasis of breast, uterine, ovarian, or pituitary cancer).Accordingly, in certain embodiments of the tumor growth inhibition ortumor regression methods provided herein, the tumor being targeted is ametastatic tumor and/or the tumor has an overexpression of ER in otherorgans (e.g., bones and/or muscles). In certain embodiments, the tumorbeing targeted is a brain tumor and/or cancer. In certain embodiments,the tumor being targeted is more sensitive to a treatment of RAD1901 anda second therapeutic agent as disclosed herein than treatment withanother SERD (e.g., fulvestrant, TAS-108 (SR16234), ZK191703, RU58668,GDC-0810 (ARN-810), GW5638/DPC974, SRN-927, ICI182782 and AZD9496), Her2inhibitors (e.g., trastuzumab, lapatinib, ado-trastuzumab emtansine,and/or pertuzumab), chemo therapy (e.g., abraxane, adriamycin,carboplatin, cytoxan, daunorubicin, doxil, ellence, fluorouracil,gemzar, helaven, lxempra, methotrexate, mitomycin, micoxantrone,navelbine, taxol, taxotere, thiotepa, vincristine, and xeloda),aromatase inhibitor (e.g., anastrozole, exemestane, and letrozole),selective estrogen receptor modulators (e.g., tamoxifen, raloxifene,lasofoxifene, and/or toremifene), angiogenesis inhibitor (e.g.,bevacizumab), and/or rituximab.

In certain embodiments of the tumor growth inhibition or tumorregression methods provided herein, the methods further comprise a stepof determining whether a patient has a tumor expressing ERα prior toadministering a combination of RAD1901 or solvates (e.g., hydrate) orsalts thereof and one or more second therapeutic agent(s) (e.g.,everolimus). In certain embodiments of the tumor growth inhibition ortumor regression methods provided herein, the methods further comprise astep of determining whether the patient has a tumor expressing mutantERα prior to administering a combination of RAD1901 or solvates (e.g.,hydrate) or salts thereof and one or more second therapeutic agent(s)(e.g., everolimus). In certain embodiments of the tumor growthinhibition or tumor regression methods provided herein, the methodsfurther comprise a step of determining whether a patient has a tumorexpressing ERα that is responsive or non-responsive to fulvestranttreatment prior to administering a combination of RAD1901 or solvates(e.g., hydrate) or salts thereof and one or more second therapeuticagent(s) (e.g., everolimus). These determinations may be made using anymethod of expression detection known in the art, and may be performed invitro using a tumor or tissue sample removed from the subject.

In addition to demonstrating the ability of RAD1901 to inhibit tumorgrowth in tumors expressing wild-type ERα, the results provided hereinshow that RAD1901 exhibited the unexpected ability to inhibit the growthof tumors expressing a mutant form of ERα, namely Y537S ERα (ExampleI(A)). Computer modeling evaluations of examples of ERα mutations showedthat none of these mutations were expected to impact the LBD orspecifically hinder RAD1901 binding (Example V(A)), e.g., ERα having oneor more mutants selected from the group consisting of ERα with Y537Xmutant wherein X is S, N, or C, ERα with D538G mutant, and ERα withS463P mutant. Based on these results, methods are provided herein forinhibiting growth or producing regression of a tumor that is positivefor ERα having one or more mutants within the ligand-binding domain(LBD), selected from the group consisting of Y537X₁ wherein X₁ is S, N,or C, D538G, L536X₂ wherein X₂ is R or Q, P535H, V534E, S463P, V3921,E380Q, especially Y537S ERα, in a subject with cancer by administeringto the subject a therapeutically effective amount of a combination ofone or more one or more second therapeutic agent(s) (e.g., everolimus)and RAD1901 or solvates (e.g., hydrate) or salts thereof. “Mutant ERα”as used herein refers to ERα comprising one or more substitutions ordeletions, and variants thereof comprising, consisting of, or consistingessentially of an amino acid sequence with at least 80%, at least 85%,at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, orat least 99.5% identity to the amino acid sequence of ERα.

In addition to inhibiting breast cancer tumor growth in an animalxenograft model, the results disclosed herein show that RAD1901 exhibitssignificant accumulation within tumor cells, and is capable ofpenetrating the blood-brain barrier (Example II). The ability topenetrate the blood-brain barrier was confirmed by showing that RAD1901administration significantly prolonged survival in a brain metastasisxenograft model (Example I(B)). Accordingly, in certain embodiments ofthe tumor growth inhibition or tumor regression methods provided herein,the ERα-positive tumor being targeted is located in the brain orelsewhere in the central nervous system. In certain of theseembodiments, the ERα-positive tumor is primarily associated with braincancer. In other embodiments, the ERα-positive tumor is a metastatictumor that is primarily associated with another type of cancer, such asbreast, uterine, ovarian, or pituitary cancer, or a tumor that hasmigrated from another tissue or organ. In certain of these embodiments,the tumor is a brain metastases, such as breast cancer brain metastases(BCBM). In certain embodiments of the methods disclosed herein, RAD1901or solvates (e.g., hydrate) or salts thereof accumulate in one or morecells within a target tumor.

In certain embodiments of the methods disclosed herein, RAD1901 orsolvates (e.g., hydrate) or salts thereof preferably accumulate in tumorat a T/P (RAD1901 concentration in tumor/RAD1901 concentration inplasma) ratio of about 15 or higher, about 18 or higher, about 19 orhigher, about 20 or higher, about 25 or higher, about 28 or higher,about 30 or higher, about 33 or higher, about 35 or higher, or about 40or higher.

The results provided herein show that RAD1901 administration protectsagainst bone loss in ovariectomized rats (Example IV(A)). Accordingly,in certain embodiments of the tumor growth inhibition or tumorregression methods provided herein, administration of a combination ofone or more second therapeutic agent(s) (e.g., everolimus) and RAD1901or solvates (e.g., hydrate) or salts thereof does not have undesirableeffects on bone, including for example undesirable effects on bonevolume density, bone surface density, bone mineral density, trabecularnumber, trabecular thickness, trabecular spacing, connectivity density,and/or apparent bone density of the treated subject. As tamoxifen may beassociated with bone loss in premenopausal women, and fulvestrant mayimpair the bone structures due to its mechanism of action, a combinationof one or more one or more second therapeutic agent(s) (e.g.,everolimus) and RAD1901 or solvates (e.g., hydrate) or salts thereof canbe particularly useful for premenopausal women, tumors resistant totamoxifen or antiestrogen therapy, and patients having osteoporosisand/or high risk of osteoporosis.

The results provided herein show that RAD1901 antagonized estradiolstimulation of uterine tissues in ovariectomized rats (Example IV(A)).Furthermore, in human subjects treated with RAD1901 at a daily dosage of200 mg or up to 500 mg, standardized uptake value (SUV) for uterus,muscle, and bone tissues that did not significantly express ER showedhardly any changes in signals pre- and post-treatment (Example III(A)).Accordingly, in certain embodiments, such administration also does notresult in undesirable effects on other tissues, including for exampleuterine, muscle, or breast tissue.

RAD1901 or solvates (e.g., hydrate) or salts thereof and the secondtherapeutic agent(s) (e.g., everolimus) are administered in combinationto a subject in need. The phrase “in combination” means that RAD1901 orsolvates (e.g., hydrate) or salts thereof may be administered before,during, or after the administration of the second therapeutic agent(s)(e.g., everolimus). For example, RAD1901 or solvates (e.g., hydrate) orsalts thereof and the second therapeutic agent(s) (e.g., everolimus) canbe administered in about one week apart, about 6 days apart, about 5days apart, about 4 days apart, about 3 days apart, about 2 days apart,about 24 hours apart, about 23 hours apart, about 22 hours apart, about21 hours apart, about 20 hours apart, about 19 hours apart, about 18hours apart, about 17 hours apart, about 16 hours apart, about 15 hoursapart, about 14 hours apart, about 13 hours apart, about 12 hours apart,about 11 hours apart, about 10 hours apart, about 9 hours apart, about 8hours apart, about 7 hours apart, about 6 hours apart, about 5 hoursapart, about 4 hours apart, about 3 hours apart, about 2 hours apart,about 1 hour apart, about 55 minutes apart, about 50 minutes apart,about 45 minutes apart, about 40 minutes apart, about 35 minutes apart,about 30 minutes apart, about 25 minutes apart, about 20 minutes apart,about 15 minutes apart, about 10 minutes apart, or about 5 minutesapart. In other embodiments RAD1901 or solvates (e.g., hydrate) or saltsthereof and the second therapeutic agent(s) (e.g., everolimus) areadministered to the subject simultaneously or substantiallysimultaneously. In certain of these embodiments, RAD1901 or solvates(e.g., hydrate) or salts thereof and the second therapeutic agent(s)(e.g., everolimus) may be administered as part of a single formulation.

In some embodiments, the combination of RAD1901 or solvates (e.g.,hydrate) or salts thereof and a single second therapeutic agent (e.g.,everolimus) is administered to a subject. In other embodiments, thecombination of RAD1901 or solvates (e.g., hydrate) or salts thereof andmore than one second therapeutic agent (e.g., everolimus) isadministered to a subject. For example, RAD1901 or solvates (e.g.,hydrate) or salts thereof can be combined with two or more secondtherapeutic agent(s) (e.g., everolimus) for treating cancers/tumors.

(2) Dosage

A therapeutically effective amount of a combination of RAD1901 orsolvates (e.g., hydrate) or salts thereof and one or more secondtherapeutic agent(s) (e.g., everolimus) for use in the methods disclosedherein is an amount that, when administered over a particular timeinterval, results in achievement of one or more therapeutic benchmarks(e.g., slowing or halting of tumor growth, resulting in tumorregression, cessation of symptoms, etc.). The combination for use in thepresently disclosed methods may be administered to a subject one time ormultiple times. In those embodiments wherein the compounds areadministered multiple times, they may be administered at a set interval,e.g., daily, every other day, weekly, or monthly. Alternatively, theycan be administered at an irregular interval, for example on anas-needed basis based on symptoms, patient health, and the like. Atherapeutically effective amount of the combination may be administereddaily for one day, at least 2 days, at least 3 days, at least 4 days, atleast 5 days, at least 6 days, at least 7 days, at least 10 days, or atleast 15 days. Optionally, the status of the cancer or the regression ofthe tumor is monitored during or after the treatment, for example, by aFES-PET scan of the subject. The dosage of the combination administeredto the subject can be increased or decreased depending on the status ofthe cancer or the regression of the tumor detected.

Ideally, the therapeutically effective amount does not exceed themaximum tolerated dosage at which 50% or more of treated subjectsexperience nausea or other toxicity reactions that prevent further drugadministrations. A therapeutically effective amount may vary for asubject depending on a variety of factors, including variety and extentof the symptoms, sex, age, body weight, or general health of thesubject, administration mode and salt or solvate type, variation insusceptibility to the drug, the specific type of the disease, and thelike.

Examples of therapeutically effective amounts of RAD1901 or solvates(e.g., hydrate) or salts thereof for use in the methods disclosed hereininclude, without limitation, about 150 to about 1,500 mg, about 200 toabout 1,500 mg, about 250 to about 1,500 mg, or about 300 to about 1,500mg daily dosage for subjects having resistant ER-driven tumors orcancers; about 150 to about 1,500 mg, about 200 to about 1,000 mg orabout 250 to about 1,000 mg or about 300 to about 1,000 mg daily dosagefor subjects having both wild-type ER driven tumors and/or cancers andresistant tumors and/or cancers; and about 300 to about 500 mg, about300 to about 550 mg, about 300 to about 600 mg, about 250 to about 500mg, about 250 to about 550 mg, about 250 to about 600 mg, about 200 toabout 500 mg, about 200 to about 550 mg, about 200 to about 600 mg,about 150 to about 500 mg, about 150 to about 550 mg, or about 150 toabout 600 mg daily dosage for subjects having majorly wild-type ERdriven tumors and/or cancers.

A therapeutically effective amount or dosage of a second therapeuticagent (e.g., everolimus) depends on its particular type. In general, thedaily dosage of a second therapeutic agent (e.g., everolimus) rangesfrom about 1 mg to about 1,500 mg, from about 1 mg to about 1,200 mg,from about 1 mg to about 1,000 mg, from about 1 mg to about 800 mg, fromabout 1 mg to about 600 mg, from about 1 mg to about 500 mg, from about1 mg to about 200 mg, from about 1 mg to about 100 mg, from about 1 mgto about 50 mg, from about 1 mg to about 30 mg, from about 1 mg to about20 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg,from about 50 mg to about 1,500 mg, from about 100 mg to about 1,200 mg,from about 150 mg to about 1,000 mg, from about 200 mg to about 800 mg,from about 300 mg to about 600 mg, from about 350 mg to about 500 mg.The daily dosage of a second therapeutic agent (e.g., everolimus) mayrange from about 1 to about 100 mg/kg, from about 1 to about 75 mg/kg,from about 1 to about 50 mg/kg, from about 1 to about 45 mg/kg, fromabout 1 to about 40 mg/kg, from about 1 to about 30 mg/kg, from about 1to about 20 mg/kg, from about 1 to about 10 mg/kg, from about 2 to about100 mg/kg, from about 2 to about 75 mg/kg, from about 2 to about 50mg/kg, from about 2 to about 45 mg/kg, from about 2 to about 40 mg/kg,from about 2 to about 30 mg/kg, from about 2 to about 20 mg/kg, fromabout 2 to about 10 mg/kg, from about 2.5 to about 100 mg/kg, from about2.5 to about 75 mg/kg, from about 2.5 to about 50 mg/kg, from about 2.5to about 45 mg/kg, from about 2.5 to about 40 mg/kg, from about 2.5 toabout 30 mg/kg, from about 2.5 to about 20 mg/kg, or from about 2.5 toabout 10 mg/kg.

In certain embodiments, a therapeutically effective amount of thecombination may utilize a therapeutically effective amount of eithercompound administered alone. In other embodiments, due to thesignificantly improved, synergistic therapeutic effect achieved by thecombination, the therapeutically effective amounts of RAD1901 orsolvates (e.g., hydrate) or salts thereof and the second therapeuticagent(s) (e.g., everolimus) when administered in the combination may besmaller than the therapeutically effective amounts of RAD1901 orsolvates (e.g., hydrate) or salts thereof and the second therapeuticagent(s) (e.g., everolimus) required when administered alone; and one orboth compounds may be administered at a dosage that is lower than thedosage at which they would normally be administered when givenseparately. Without being bound by any specific theory, the combinationtherapy achieves a significantly improved effect by reducing the dosageof at least one or all of RAD1901 or solvates (e.g., hydrate) or saltsthereof and the second therapeutic agent(s) (e.g., everolimus), therebyeliminating or alleviating undesirable toxic side effects.

In some embodiments, the therapeutically effective amount of RAD1901 orsolvates (e.g., hydrate) or salts thereof when administered as part ofthe combination is about 30% to about 200%, about 40% to about 200%,about 50% to about 200%, about 60% to about 200%, about 70% to about200%, about 80% to about 200%, about 90% to about 200%, about 100% toabout 200%, 30% to about 150%, about 40% to about 150%, about 50% toabout 150%, about 60% to about 150%, about 70% to about 150%, about 80%to about 150%, about 90% to about 150%, about 100% to about 150%, about30% to about 120%, about 40% to about 120%, about 50% to about 120%,about 60% to about 120%, about 70% to about 120%, about 80% to about120%, about 90% to about 120%, about 100% to about 120%, 30% to about110%, about 40% to about 110%, about 50% to about 110%, about 60% toabout 110%, about 70% to about 110%, about 80% to about 110%, about 90%to about 110%, or about 100% to about 110% of the therapeuticallyeffective amount of RAD1901 or solvates (e.g., hydrate) or salts thereofwhen administered alone. In some embodiments, the therapeuticallyeffective amount of the second therapeutic agent(s) (e.g., everolimus)when administered as part of the combination is about 30% to about 200%,about 40% to about 200%, about 50% to about 200%, about 60% to about200%, about 70% to about 200%, about 80% to about 200%, about 90% toabout 200%, about 100% to about 200%, 30% to about 150%, about 40% toabout 150%, about 50% to about 150%, about 60% to about 150%, about 70%to about 150%, about 80% to about 150%, about 90% to about 150%, about100% to about 150%, about 30% to about 120%, about 40% to about 120%,about 50% to about 120%, about 60% to about 120%, about 70% to about120%, about 80% to about 120%, about 90% to about 120%, about 100% toabout 120%, 30% to about 110%, about 40% to about 110%, about 50% toabout 110%, about 60% to about 110%, about 70% to about 110%, about 80%to about 110%, about 90% to about 110%, or about 100% to about 110% ofthe therapeutically effective amount of the second therapeutic agent(s)(e.g., everolimus) when administered alone.

In certain embodiments, the cancers or tumors are resistant ER-drivencancers or tumors (e.g. having mutant ER binding domains (e.g. ERαcomprising one or more mutations including, but not limited to, Y537X₁wherein X₁ is S, N, or C, D538G, L536X₂ wherein X₂ is R or Q, P535H,V534E, S463P, V392I, E380Q and combinations thereof), overexpressors ofthe ERs or tumor and/or cancer proliferation becomes ligand independent,or tumors and/or cancers that progress with treatment of another SERD(e.g., fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810(ARN-810), GW5638/DPC974, SRN-927, ICI182782 and AZD9496), Her2inhibitors (e.g., trastuzumab, lapatinib, ado-trastuzumab emtansine,and/or pertuzumab), chemo therapy (e.g., abraxane, adriamycin,carboplatin, cytoxan, daunorubicin, doxil, ellence, fluorouracil,gemzar, helaven, lxempra, methotrexate, mitomycin, micoxantrone,navelbine, taxol, taxotere, thiotepa, vincristine, and xeloda),aromatase inhibitor (e.g., anastrozole, exemestane, and letrozole),selective estrogen receptor modulators (e.g., tamoxifen, raloxifene,lasofoxifene, and/or toremifene), angiogenesis inhibitor (e.g.,bevacizumab), and/or rituximab.

In certain embodiments, the dosage of RAD1901 or solvates (e.g.,hydrate) or salts thereof in a combination with a second therapeuticagent (e.g., everolimus) as described herein (e.g., ribociclib,abemaciclib and everolimus) for use in the presently disclosed methodsgeneral for an adult subject may be approximately 30 μg to 2,000 mg, 100μg to 1,500 mg, or 150 mg to 1,500 mg per day via oral administration.This daily dosage may be achieved via a single administration ormultiple administrations.

A combination of one or more second therapeutic agent(s) (e.g.,everolimus) and RAD1901 or solvates (e.g., hydrate) or salts thereof maybe administered to a subject one time or multiple times. In thoseembodiments wherein the compounds are administered multiple times, theymay be administered at a set interval, e.g., daily, every other day,weekly, or monthly. Alternatively, they can be administered at anirregular interval, for example on an as-needed basis based on symptoms,patient health, and the like. (3) Formulation

In some embodiments, RAD1901 or solvates (e.g., hydrate) or saltsthereof and the second therapeutic agent(s) (e.g., everolimus) areadministered in separate formulations. In certain of these embodiments,the formulations may be of the same type. For example, both formulationsmay be designed for oral administration (e.g., via two separate pills)or for injection (e.g., via two separate injectable formulations). Inother embodiments, RAD1901 or solvates (e.g., hydrate) or salts thereofand the second therapeutic agent(s) (e.g., everolimus) may be formulatedin different types of formulations. For example, one compound may be ina formulation designed for oral administration, while the other is in aformulation designed for injection.

In other embodiments, RAD1901 or solvates (e.g., hydrate) or saltsthereof and the second therapeutic agent(s) (e.g., everolimus) describedherein are administered as part of a single formulation. For example,RAD1901 or solvates (e.g., hydrate) or salts thereof and the secondtherapeutic agent(s) (e.g., everolimus) are formulated in a single pillfor oral administration or in a single dose for injection. Providedherein in certain embodiments are combination formulations comprisingRAD1901 or solvates (e.g., hydrate) or salts thereof and one or moresecond therapeutic agent(s) (e.g., everolimus). In certain embodiments,administration of the compounds in a single formulation improves patientcompliance.

The therapeutically effective amount of each compound when administeredin combination may be lower than the therapeutically effective amount ofeach compound administered alone.

In some embodiments, a formulation comprising RAD1901 or solvates (e.g.,hydrate) or salts thereof, one or more to the second therapeuticagent(s) (e.g., everolimus), or both RAD 1901 or solvates (e.g.,hydrate) or salts thereof and the one or more second therapeuticagent(s) (e.g., everolimus) may further comprise one or morepharmaceutical excipients, carriers, adjuvants, and/or preservatives.

RAD1901 or solvates (e.g., hydrate) or salts thereof and the secondtherapeutic agent(s) (e.g., everolimus) for use in the presentlydisclosed methods can be formulated into unit dosage forms, meaningphysically discrete units suitable as unitary dosage for subjectsundergoing treatment, with each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effect,optionally in association with a suitable pharmaceutical carrier. Theunit dosage form can be for a single daily dose or one of multiple dailydoses (e.g., about 1 to 4 or more times per day). When multiple dailydoses are used, the unit dosage form can be the same or different foreach dose. In certain embodiments, the compounds may be formulated forcontrolled release.

RAD1901 or solvates (e.g., hydrate) or salts thereof and the secondtherapeutic agent(s) (e.g., everolimus) for use in the presentlydisclosed methods can be formulated according to any availableconventional method. Examples of preferred dosage forms include atablet, a powder, a subtle granule, a granule, a coated tablet, acapsule, a syrup, a troche, an inhalant, a suppository, an injectable,an ointment, an ophthalmic ointment, an eye drop, a nasal drop, an eardrop, a cataplasm, a lotion and the like. In the formulation, generallyused additives such as a diluent, a binder, an disintegrant, alubricant, a colorant, a flavoring agent, and if necessary, astabilizer, an emulsifier, an absorption enhancer, a surfactant, a pHadjuster, an antiseptic, an antioxidant and the like can be used. Inaddition, the formulation is also carried out by combining compositionsthat are generally used as a raw material for pharmaceuticalformulation, according to the conventional methods. Examples of thesecompositions include, for example, (1) an oil such as a soybean oil, abeef tallow and synthetic glyceride; (2) hydrocarbon such as liquidparaffin, squalane and solid paraffin; (3) ester oil such asoctyldodecyl myristic acid and isopropyl myristic acid; (4) higheralcohol such as cetostearyl alcohol and behenyl alcohol; (5) a siliconresin; (6) a silicon oil; (7) a surfactant such as polyoxyethylene fattyacid ester, sorbitan fatty acid ester, glycerin fatty acid ester,polyoxyethylene sorbitan fatty acid ester, a solid polyoxyethylenecastor oil and polyoxyethylene polyoxypropylene block co-polymer; (8)water soluble macromolecule such as hydroxyethyl cellulose, polyacrylicacid, carboxyvinyl polymer, polyethyleneglycol, polyvinylpyrrolidone andmethylcellulose; (9) lower alcohol such as ethanol and isopropanol; (10)multivalent alcohol such as glycerin, propyleneglycol, dipropyleneglycoland sorbitol; (11) a sugar such as glucose and cane sugar; (12) aninorganic powder such as anhydrous silicic acid, aluminum magnesiumsilicicate and aluminum silicate; (13) purified water, and the like.Additives for use in the above formulations may include, for example, 1)lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystallinecellulose and silicon dioxide as the diluent; 2) polyvinyl alcohol,polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic,tragacanth, gelatine, shellac, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polypropyleneglycol-poly oxyethylene-block co-polymer, meglumine, calcium citrate,dextrin, pectin and the like as the binder; 3) starch, agar, gelatinepowder, crystalline cellulose, calcium carbonate, sodium bicarbonate,calcium citrate, dextrin, pectic, carboxymethylcellulose/calcium and thelike as the disintegrant; 4) magnesium stearate, talc,polyethyleneglycol, silica, condensed plant oil and the like as thelubricant; 5) any colorants whose addition is pharmaceuticallyacceptable is adequate as the colorant; 6) cocoa powder, menthol,aromatizer, peppermint oil, cinnamon powder as the flavoring agent; 7)antioxidants whose addition is pharmaceutically accepted such asascorbic acid or alpha-tophenol.

RAD1901 or solvates (e.g., hydrate) or salts thereof and one or moresecond therapeutic agent(s) (e.g., everolimus) for use in the presentlydisclosed methods can be formulated into a pharmaceutical composition asany one or more of the active compounds described herein and aphysiologically acceptable carrier (also referred to as apharmaceutically acceptable carrier or solution or diluent). Suchcarriers and solutions include pharmaceutically acceptable salts andsolvates of compounds used in the methods of the instant invention, andmixtures comprising two or more of such compounds, pharmaceuticallyacceptable salts of the compounds and pharmaceutically acceptablesolvates of the compounds. Such compositions are prepared in accordancewith acceptable pharmaceutical procedures such as described inRemington's Pharmaceutical Sciences, 18th edition, ed. Alfonso R.Gennaro, Mack Printing Company, Eaton, Pa. (1990), which is incorporatedherein by reference.

The term “pharmaceutically acceptable carrier” refers to a carrier thatdoes not cause an allergic reaction or other untoward effect in patientsto whom it is administered and are compatible with the other ingredientsin the formulation. Pharmaceutically acceptable carriers include, forexample, pharmaceutical diluents, excipients or carriers suitablyselected with respect to the intended form of administration, andconsistent with conventional pharmaceutical practices. For example,solid carriers/diluents include, but are not limited to, a gum, a starch(e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose,mannitol, sucrose, dextrose), a cellulosic material (e.g.,microcrystalline cellulose), an acrylate (e.g., polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the therapeutic agent.

The one or more second therapeutic agent(s) (e.g., everolimus) andRAD1901 or solvates (e.g., hydrate) or salts thereof in a free form canbe converted into a salt by conventional methods. The term “salt” usedherein is not limited as long as the salt is formed with RAD1901 orsolvates (e.g., hydrate) or salts thereof and is pharmacologicallyacceptable; preferred examples of salts include a hydrohalide salt (forinstance, hydrochloride, hydrobromide, hydroiodide and the like), aninorganic acid salt (for instance, sulfate, nitrate, perchlorate,phosphate, carbonate, bicarbonate and the like), an organic carboxylatesalt (for instance, acetate salt, maleate salt, tartrate salt, fumaratesalt, citrate salt and the like), an organic sulfonate salt (forinstance, methanesulfonate salt, ethanesulfonate salt, benzenesulfonatesalt, toluenesulfonate salt, camphorsulfonate salt and the like), anamino acid salt (for instance, aspartate salt, glutamate salt and thelike), a quaternary ammonium salt, an alkaline metal salt (for instance,sodium salt, potassium salt and the like), an alkaline earth metal salt(magnesium salt, calcium salt and the like) and the like. In addition,hydrochloride salt, sulfate salt, methanesulfonate salt, acetate saltand the like are preferred as “pharmacologically acceptable salt” of thecompounds according to the present invention.

Isomers of RAD1901 or solvates (e.g., hydrate) or salts thereof and/orthe second therapeutic agent(s) (e.g., everolimus) (e.g., geometricisomers, optical isomers, rotamers, tautomers, and the like) can bepurified using general separation means, including for examplerecrystallization, optical resolution such as diastereomeric saltmethod, enzyme fractionation method, various chromatographies (forinstance, thin layer chromatography, column chromatography, glasschromatography and the like) into a single isomer. The term “a singleisomer” herein includes not only an isomer having a purity of 100%, butalso an isomer containing an isomer other than the target, which existseven through the conventional purification operation. A crystalpolymorph sometimes exists for RAD1901 or solvates (e.g., hydrate) orsalts thereof and/or a second therapeutic agent (e.g., everolimus), andall crystal polymorphs thereof are included in the present invention.The crystal polymorph is sometimes single and sometimes a mixture, andboth are included herein.

In certain embodiments, RAD1901 or solvates (e.g., hydrate) or saltsthereof and/or second therapeutic agent (e.g., everolimus) may be in aprodrug form, meaning that it must undergo some alteration (e.g.,oxidation or hydrolysis) to achieve its active form. Alternative,RAD1901 or solvates (e.g., hydrate) or salts thereof and/or secondtherapeutic agent (e.g., everolimus) may be a compound generated byalteration of a parental prodrug to its active form.

(4) Administration Route

Administration routes of RAD1901 or solvates (e.g., hydrate) or saltsthereof and/or second therapeutic agent(s) (e.g., everolimus) disclosedherein include but not limited to topical administration, oraladministration, intradermal administration, intramuscularadministration, intraperitoneal administration, intravenousadministration, intravesical infusion, subcutaneous administration,transdermal administration, and transmucosal administration.

(5) Gene Profiling

In certain embodiments, the methods of tumor growth inhibition or tumorregression provided herein further comprise gene profiling the subject,wherein the gene to be profiled is one or more genes selected from thegroup consisting of ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM,AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3,CCNE1, CDH1, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA,CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1,EWSR1, FBXW7, FGF4, FGFR1, FGFR2, FGFR3, FLT3, FRS2, HIF1A, HRAS, IDH1,IDH2, IGF1R, JAK2, KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4,MCL1, MDM2, MDM4, MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2,NKX2-1, NOTCH1, NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD,RARA, RB1, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11,TET2, TP53, TSC1, TSC2, and VHL.

In certain embodiments, the second agent is everolimus, and subjectspresent somatic mutations in TSC1.

In some embodiments, this invention provides a method of treating asubpopulation of breast cancer patients wherein said sub-population hasincreased expression of one or more of the following genes and treatingsaid sub-population with an effective dose of a combination of RAD1901or solvates (e.g., hydrate) or salts thereof and one or more secondtherapeutic agent(s) (e.g., everolimus) as described herein according tothe dosing embodiments as described in this disclosure.

(6) Dose Adjusting

In addition to establishing the ability of RAD1901 to inhibit tumorgrowth, the results provided herein show that RAD1901 inhibits estradiolbinding to ER in the uterus and pituitary (Example III(A)). In theseexperiments, estradiol binding to ER in uterine and pituitary tissue wasevaluated by FES-PET imaging. After treatment with RAD1901, the observedlevel of ER binding was at or below background levels. These resultsestablish that the antagonistic effect of RAD1901 on ER activity can beevaluated using real-time scanning. Based on these results, methods areprovided herein for monitoring the efficacy of treatment RAD1901 orsolvates (e.g., hydrate) or salts thereof in a combination therapydisclosed herein by measuring estradiol-ER binding in one or more targettissues, wherein a decrease or disappearance in binding indicatesefficacy.

Further provided are methods of adjusting the dosage of RAD1901 orsolvates (e.g., hydrate) or salts thereof in a combination therapydisclosed herein based on estradiol-ER binding. In certain embodimentsof these methods, binding is measured at some point following one ormore administrations of a first dosage of the compound. If estradiol-ERbinding is not affected or exhibits a decrease below a predeterminedthreshold (e.g., a decrease in binding versus baseline of less than 5%,less than 10%, less than 20%, less than 30%, or less than 50%), thefirst dosage is deemed to be too low. In certain embodiments, thesemethods comprise an additional step of administering an increased seconddosage of the compound. These steps can be repeated, with dosagerepeatedly increased until the desired reduction in estradiol-ER bindingis achieved. In certain embodiments, these steps can be incorporatedinto the methods of inhibiting tumor growth provided herein. In thesemethods, estradiol-ER binding can serve as a proxy for tumor growthinhibition, or a supplemental means of evaluating growth inhibition. Inother embodiments, these methods can be used in conjunction with theadministration of RAD1901 or solvates (e.g., hydrate) or salts thereoffor purposes other than inhibition of tumor growth, including forexample inhibition of cancer cell proliferation.

In certain embodiments, the methods provided herein for adjusting thedosage of a RAD1901 or solvates (e.g., hydrate) or salts thereof in acombination therapy comprise:

-   (1) administering a first dosage of RAD1901 or solvates (e.g.,    hydrate) or salts thereof (e.g., about 350 to about 500 mg/day) for    3, 4, 5, 6, or 7 days;-   (2) detecting estradiol-ER binding activity, for example using    FES-PET imaging as disclosed herein; wherein:    -   (i) if the ER binding activity is not detectable or is below a        predetermined threshold level, continuing to administer the        first dosage (i.e., maintain the dosage level); or    -   (ii) if the ER binding activity is detectable or is above a        predetermined threshold level, administering a second dosage        that is greater than the first dosage (e.g., the first dosage        plus about 50 to about 200 mg) for 3, 4, 5, 6, or 7 days, then        proceeding to step (3);-   (3) detecting estradiol-ER binding activity, for example using    FES-PET imaging as disclosed herein; wherein    -   (i) if the ER binding activity is not detectable or is below a        predetermined threshold level, continuing to administer the        second dosage (i.e., maintain the dosage level); or    -   (ii) if the ER binding activity is detectable or is above a        predetermined threshold level, administering a third dosage that        is greater than the second dosage (e.g., the second dosage plus        about 50 to about 200 mg) for 3, 4, 5, 6, or 7 days, then        proceeding to step (4);-   (4) repeating the steps above through a fourth dosage, fifth dosage,    etc., until no ER binding activity is detected.

In certain embodiments, the invention includes the use of PET imaging todetect and/or dose ER sensitive or ER resistant cancers.

(7) Combinations for the Methods Disclosed Herein

Another aspect of the invention relates to a pharmaceutical compositioncomprising one or more RAD1901 or solvates (e.g., hydrate) or saltsthereof and/or second therapeutic agent(s) (e.g., everolimus) disclosedherein in a therapeutically effective amount as disclosed herein for thecombination methods set forth herein.

RAD1901-ERα Interactions

(1) Mutant ERα in ER Positive Breast Tumor Samples from Patients WhoReceived at Least One Line of Endocrine Treatment

In five studies reported in the past two years, a total of 187metastatic ER positive breast tumor samples from patients who receivedat least one line of endocrine treatment were sequenced and ER LBDmutations were identified in 39 patients (21%) (Jeselsohn). Among the 39patients, the six most frequent LBD mutations are shown in Scheme 1adapted from Jeselsohn.

The frequency of all LBD mutations are summarized in Table 9.

Computer modeling showed that RAD1901-ERα interactions are not likely tobe affected by mutants of LBD of ERα, e.g., Y537X mutant wherein X wasS, N, or C; D538G; and S463P, which account for about 81.7% of LBDmutations found in a recent study of metastatic ER positive breast tumorsamples from patients who received at least one line of endocrinetreatment (Table 10, Example V).

Provided herein are complexes and crystals of RAD1901 bound to ERαand/or a mutant ERα, the mutant ERα comprises one or more mutationsincluding, but not limited to, Y537X₁ wherein X₁ is S, N, or C, D538G,L536X₂ wherein X₂ is R or Q, P535H, V534E, S463P, V392I, E380Q andcombinations thereof.

In certain embodiments of the methods provided herein, the LBD of ERαand a mutant ERα comprises AF-2. In other embodiments, the LBDcomprises, consists of, or consists essentially of amino acids 299-554of ERα. In certain embodiments, the LBD of the mutant ERα comprises oneor more mutations including, but not limited to, Y537X₁ wherein X₁ is S,N, or C, D538G, L536X₂ wherein X₂ is R or Q, P535H, V534E, S463P, V392I,E380Q and combinations thereof. The term “and/or” as used hereinincludes both the “and” case and the “or” case.

Provided herein in certain embodiments are methods of treating acondition associated with ERα and/or a mutant ERα activity or expressionin a subject in need thereof comprising administering to the subject acombination of one or more second therapeutic agent(s) (e.g.,everolimus) and one or more compounds capable of binding to ERα and/or amutant ERα via LBD. In certain embodiments, the subject is a mammal, andin certain of these embodiments the subject is human. In certainembodiments, the condition is tumor and/or cancer, including but notlimited to ER positive tumor and/or cancer as disclosed herein.

In certain embodiments of the compounds and methods provided herein, theLBD of ERα and a mutant ERα comprises AF-2. In other embodiments, theLBD comprises, consists of, or consists essentially of amino acids299-554 of ERα. In certain embodiments, the LBD of the mutant ERαcomprises one or more mutations including, but not limited to, Y537X1wherein X₁ is S, N, or C, D538G, L536X₂ wherein X₂ is R or Q, P535H,V534E, S463P, V392I, E380Q and combinations thereof.

In certain embodiments of the compounds and methods provided herein, thecompound capable of binding to ERα and/or mutant ERα via LBD is aselective estrogen receptor degrader (SERD) or selective estrogenreceptor modulator (SERM). In certain embodiments, the compound capableof binding to ERα and/or mutant ERα via LBD does so via one or moreinteractions selected from the group consisting of H-bond interactionswith residues E353, D351, R349, and/or L536 and pi-interactions withresidue F404 of ERα and/or mutant ERα. One example of such a compound isRAD1901.

Provided herein in certain embodiments are methods of treating acondition associated with activity or expression of a mutant ERαcomprising one or more mutations including, but not limited to, Y537X₁wherein X₁ is S, N, or C, D538G, L536X₂ wherein X2 is R or Q, P535H,V534E, S463P, V3921, E380Q and combinations thereof, wherein the methodcomprises administering to the subject a combination of one or moresecond therapeutic agent(s) (e.g., everolimus) and one or more compoundscapable of binding to ERα via the LBD. In certain embodiments, thecondition is cancer, including but not limited to ER positive cancer,breast cancer, ER positive breast cancer, and metastatic breast cancer,and in certain embodiments the compound is RAD1901 or a pharmaceuticallyacceptable solvate (e.g., hydrate) or pharmaceutically acceptable saltthereof.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention. It will be understood thatmany variations can be made in the procedures herein described whilestill remaining within the bounds of the present invention. It is theintention of the inventors that such variations are included within thescope of the invention.

EXAMPLES Materials and Methods Test Compounds

RAD1901 used in the examples below was(6R)-6-(2-(N-(4-(2-(ethylamino)ethyl)benzyl)-N-ethylamino)-4-methoxyphenyl)-5,6,7,8-tetrahydronaphthalen-2-oldihydrochloride, manufactured by IRIX Pharmaceuticals, Inc. (Florence,S.C.). RAD1901 was stored as a dry powder, formulated for use as ahomogenous suspension in 0.5% (w/v) methylcellulose in deionized water,and for animal models was administered by oral gavage. Tamoxifen,raloxifene and estradiol (E2) were obtained from Sigma-Aldrich (St.Louis, Mo.), and administered by subcutaneous injection. Fulvestrant wasobtained from Tocris Biosciences (Minneapolis, Minn.) and administeredby subcutaneous injection. Other laboratory reagents were purchased fromSigma-Aldrich unless otherwise noted.

Cell Lines

MCF-7 cells (human mammary metastatic adenocarcinoma) were purchasedfrom American Type Culture Collection (Rockville, Md.) and wereroutinely maintained in phenol red-free minimal essential medium (MEM)containing 2 mM L-glutamine and Earle's BSS, 0.1 mM non-essential aminoacids and 1 mM sodium pyruvate supplemented with 0.01 mg/ml bovineinsulin and 10% fetal bovine serum (Invitrogen, Carlsbad, Calif.), at 5%CO₂.

T47D cells were cultured in 5% CO₂ incubator in 10 cm dishes toapproximately 75% confluence in RPMI growth media supplemented with 10%FBS and 5 μg/mL human insulin.

In vivo Xenograft Models

All mice were housed in pathogen-free housing in individually ventilatedcages with sterilized and dust-free bedding cobs, access to sterilizedfood and water ad libitum, under a light dark cycle (12-14 hourcircadian cycle of artificial light) and controlled room temperature andhumidity. Tumors were measured twice weekly with Vernier calipers andvolumes were calculated using the formula: (L*W²)*0.52.

PDx Models

Some examples of patient-derived xenograft models (PDx models) are shownin FIG. 1. PDx models with patient derived breast cancer tumor wereestablished from viable human tumor tissue or fluid that had beenserially passaged in animals (athymic nude mice (Nu (NCF)-Foxn1nu)) alimited number of times to maintain tumor heterogeneity. Pre-study tumorvolumes were recorded for each experiment beginning approximately oneweek prior to its estimated start date. When tumors reached theappropriate Tumor Volume Initiation (TVI) range (150-250 mm³), animalswere randomized into treatment and control groups and dosing initiated(Day 0, 8-10 subjects in each group); animals in all studies followedindividually throughout each experiment. Initial dosing began Day 0;animals in all groups were dosed by weight (0.01 mL per gram; 10 ml/kg).Each group was treated with vehicle (control, p.o./QD to the endpoint),tamoxifen (1 mg/subject, s.c./QOD to the end point), fulvestrant(Faslodex®; 1 mg/subject or 3 mg/subject as needed, SC/weekly X 5 andextended if necessary), or RAD1901 (30, 60 or 120 mg/kg of the subject,p.o./QD to the endpoint) as specified from day 0. The treatment periodlasted for 56-60 days depending on the models. The drinking water forthese PDx models was supplemented with 17β-estradiol.

Agent Efficacy

For all studies, beginning Day 0, tumor dimensions were measured bydigital caliper and data including individual and mean estimated tumorvolumes (Mean TV±SEM) recorded for each group; tumor volume wascalculated using the formula (Yasui et al. Invasion Metastasis17:259-269 (1997), which is incorporated herein by reference):TV=width²×length×0.52. Each group or study was ended once the estimatedgroup mean tumor volume reached the Tumor Volume (TV) endpoint (timeendpoint was 60 days; and volume endpoint was group mean 2 cm³);individual mice reaching a tumor volume of 2 cm³ or more were removedfrom the study and the final measurement included in the group meanuntil the mean reached volume endpoint or the study reached timeendpoint.

Efficacy Calculations and Statistical Analysis

% Tumor Growth Inhibition (% TGI) values were calculated at a singletime point (when the control group reached tumor volume or timeendpoint) and reported for each treatment group (T) versus control (C)using initial (i) and final (f) tumor measurements by the formula(Corbett TH et al. In vivo methods for screening and preclinicaltesting. In: Teicher B, ed., Anticancer Drug Development Guide. Totowa,N.J.: Humana. 2004: 99-123.): %TGI=1−Tf−Ti/Cf−Ci.

Statistics

TGI Studies-One way ANOVA+Dunnett's Multiple Comparisons Test (Corbett TH et al).

Sample Collection

At endpoint, tumors were removed. One fragment was flash frozen, whileanother fragment was placed in 10% NBF for at least 24 hours andformalin fixed paraffin embedded (FFPE). Flash frozen samples werestored at −80° C.; FFPE blocks were stored at room temperature.

Western Blot

Cells were harvested and protein expression was analyzed using standardpractice. Tumors were harvested at the indicated time points after thelast day of dosing, homogenized in RIPA buffer with protease andphosphatase inhibitors using a Tissuelyser (Qiagen). Equal amounts ofprotein were separated by MW, transferred to nitrocellulose membranesand blotted with the following antibodies using standard practice:

-   -   Estrogen receptor (Santa Cruz (HC-20); sc-543)    -   Progesterone receptor (Cell Signaling Technologies; 3153)    -   Vinculin (Sigma-Aldrich, v9131)

qPCR analyses were performed as follows: Cells were harvested, mRNA wasextracted, and equal amounts used for cDNA synthesis and qPCR withprimers specific for progesterone receptor, GREB1, and TFF1 (LifeTech).Bands were quantified using 1D Quant software (GE).

Immunohistochemistry

Tumors were harvested, formalin-fixed and embedded into paraffin.Embedded tumors were sectioned (6 μM) and stained with antibodiesspecific for ER, PR, and Her2. Quantitation was performed as follows:Five fields were counted for positive cells (0-100%) and intensity ofstaining (0-3+). H-scores (0-300) were calculated using the followingformula: % positivity*intensity.

Example I RAD1901-Everolimus Combinations Provided Enhanced Tumor GrowthInhibition in Tumor and/or Cancer Expressing WT ER or Mutant ER (e.g.,Y537S), with Different Prior Endocrine Therapy

I(A). Effectiveness of RAD1901 on animal xenografts models

I(A)(i) RAD1901 inhibited tumor growth in PDx models (PDx-1 to PDx-12)regardless of ER status and prior endocrine therapy

FIG. 1 demonstrates tumor growth inhibition effects in various PDxmodels for mice treated with RAD1901 alone. Twelve patient-derivedxenograft models were screened to test RAD1901 response in a variety ofgenetic backgrounds with varied levels of ER, PR and Her2. Full efficacystudy was carried out for PDx models marked with “*” (PDx-1 to PDx-4,and PDx-12), with n=8-10. Screen study was carried out for other PDxmodels (PDx-5 to PDx-11), with n=3. The PDx models were treated withvehicle (negative control) or RAD1901 at a dosage of 60 mg/kg for 60days p.o., q.d. As demonstrated in FIG. 1, PDx models in which thegrowth was driven by ER and an additional driver (e.g., PR+ and/orHer2+) benefited from the RAD1901 treatments. RAD1901 was efficacious ininhibiting tumor growth in models with ER mutations and/or high levelexpression of Her2 (PDx), regardless of prior treatment, eithertreatment naive (Rx-naive), or treated with aromatase inhibitor,tamoxifen (tam), chemotherapy (chemo), Her2 inhibitors (Her2i, e.g.,trastuzumab, lapatinib), bevacizumab, fulvestrant, and/or rituximab.

I(A)(ii) RAD1901-everolimus combination drove more regression thanRAD1901 alone in xenograft models expressing WT ER

1(A)(ii)(1) RAD1901-everolimus drove more regression than RAD1901 alonein MCF-7 xenografts that were responsive to fulvestrant treatments.

MCF-7 Xenograft Model

Two days before cell implantation, Balb/C-Nude mice were inoculated with0.18/90-day release 17β-estradiol pellets. MCF-7 cells (PR+, Her2-) wereharvested and 1×10⁷ cells were implanted subcutaneously in the rightflank of Balb/C-Nude mice. When the tumors reached an average of 200mm³, the mice were randomized into treatment groups by tumor volume andtreated with the test compounds. Each group was treated with vehicle(control, p.o., q.d., to the endpoint), fulvestrant (Faslodex®; 3mg/subject, s.c., qwk X 5 and extended if necessary), RAD1901 (30 mg/kgor 60 mg/kg of the subject, p.o., q.d., to the endpoint), everolimus(2.5 mg/kg, p.o., to the end point), or RAD1901-everolimus combinationat doses specified from day 0. The treatment period lasted for 28 days.

MCF-7 xenograft mice were treated with vehicle (negative control),RAD1901 (60 mg/kg, PO daily), everolimus (2.5 mg/kg, p.o.), acombination of RAD1901 (30 or 60 mg/kg, PO daily) and everolimus (2.5mg/kg, p.o.), fulvestrant (3 mg/dose, s.c., weekly) or a combination offulvestrant (3 mg/dose, s.c., weekly) and everolimus (2.5 mg/kg, p.o.).Tumor size was measured at various time points for 27 days.

Results are shown in FIGS. 2A-2B. Treatment with the combination ofRAD1901 (60 mg/kg) and everolimus (2.5 mg/kg), once again resulted insignificant tumor regression, with superior results to treatment withRAD1901, everolimus, or fulvestrant alone, or with a combination offulvestrant and everolimus (FIG. 2B).

FIG. 2C demonstrates that RAD1901-everolimus combinations with RAD1901at a dose of 30 mg/kg or 60 mg/kg both provided similar effects,although RAD1901 alone at 30 mg/kg was not as effective as RAD1901 aloneat 60 mg/kg in inhibiting tumor growth. Said results suggest aRAD1901-everolimus combination with a lower dose of RAD1901(e.g., 30mg/kg) was sufficient to maximize the tumor growth inhibition/tumorregression effects in said xenograft models.

Treatment with the combination of RAD1901 and everolimus was also moreeffective at decreasing ER and PR expression in vivo in the MCF-7xenograft models than treatment with RAD1901, everolimus, or fulvestrantalone, or treatment with a combination of fulvestrant and everolimus(FIG. 11); tumors harvested two hours after the last dosing).

1(A)(ii)(2) RAD1901-everolimus drove more regression than RAD1901 alonein PDx-11 and PDx-2 models that were responsive to fulvestranttreatments.

ER WT PDx models PDx-2 (PR+, Her2+, treatment naïve) and PDx-11 (PR+,Her2+, treated with AI, fulvestrant and chemo) exhibited differentsensitivities to fulvestrant (3 mg/dose, qwk, s.c.). PDx-2 and PDx-11models were treated with a combination of RAD1901 (60 mg/kg, q.d., p.o.)and everolimus (2.5 mg/kg, p.o.), RAD1901 alone (60 mg/kg, q.d., p.o.),everolimus alone (2.5 mg/kg, p.o.), or fulvestrant alone (3 mg/dose,qwk, s.c.).

In PDx-11 models, administration of fulvestrant or everolimus alonesignificantly inhibited tumor growth, with fulvestrant treated miceexhibiting better effects in tumor growth inhibition. Fulvestranttreatment exhibited slight tumor regression (FIG. 3B). Unexpectedly,administration of RAD1901 alone or in combination with everolimusresulted in a significant tumor regression, with the combinationachieved even more significant tumor regression effects in the wild-typeESR1 PDx models (FIG. 3B).

In PDx-2 models, oral administration of RAD1901 alone achieved bettereffects of inhibiting tumor growth comparing to injection of fulvestrantalone (FIG. 4A). Furthermore, administration of RAD1901 or everolimusalone significantly inhibited tumor growth. Unexpectedly, administrationof RAD1901 in combination with everolimus resulted in even more enhancedeffect in inhibiting tumor growth (FIG. 4B).

Furthermore, in PDx-4 model that were responsive to fulvestranttreatment (1 mg/dose, s.c., qwk), RAD1901-mediated tumor growthinhibition was maintained in the absence of treatment at least twomonths after RAD1901 treatment (30 mg/kg, p.o., q.d.) period ended,while estradiol treatment continued (FIG. 5).

Thus, a combination of one or more second therapeutic agent (s) withRAD1901 is likely to benefit a patient in inhibiting tumor growth aftertreatment ends, especially when the one or more second therapeutic agent(s) (e.g., everolimus) can be reduced or delayed for adverse reactions.http://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm488028.htm.

1(A)(iii) RAD1901-everolimus drove more regression than RAD1901 alone inxenograft models expressing mutant ER (ERα Y537S)

1(A)(iii)(1) RAD1901-everolimus drove more regression than RAD1901 alonein PDx-5 models that were hardly responsive to fulvestrant treatments.

PDx-5 models were prepared following similar protocol as described suprafor PDx models. The tumor sizes of each dosing group were measured twiceweekly with Vernier calipers, and volumes were calculated using theformula (L*W2)*0.52.

Inhibition of tumor growth by RAD1901 (60 mg/kg, q.d., p.o.), everolimus(2.5 mg/kg, p.o.), and RAD1901 (60 mg/kg, q.d., p.o.) in combinationwith everolimus (2.5 mg/kg, p.o.) in PDx-5 models (PDx models withpatient-derived breast cancer tumor having the Y537S estrogen receptormutation, PR+, Her2+, prior treatment with aromatase inhibitor) wasassessed using the method described herein. For tumors expressingcertain ERα mutations (e.g., Y537S), combination treatment of RAD1901and everolimus was more effective in inhibiting tumor growth thantreatment with either agent alone (FIG. 6B). These PDx models werehardly responsive to fulvestrant (3 mg/dose). Combination treatment ofRAD1901 and everolimus was more effective than treatment with eitheragent alone in inhibiting tumor growth in the PDx-5 models (FIG. 6B).

Thus, the results showed that RAD1901 was an effective endocrinebackbone that potentiated the tumor growth inhibition of targetedagents. Furthermore, RAD1901 showed potent anti-tumor activity in PDxmodels derived from patients that have had multiple prior endocrinetherapies including those that are insensitive to fulvestrant.

I(A) (iv) Pharmacokinetic evaluation of fulvestrant treatments tonon-tumor bearing mice.

Various doses of fulvestrant were administered to mice and demonstratedsignificant dose exposure to the subjects (FIG. 7).

Fulvestrant was administered at 1, 3, or 5 mg/dose subcutaneously tonude mice on day 1 (D1 Rx) and day 8 (D8 Rx, n=4/dose level). Blood wascollected at the indicated time points for up to 168 hours after thesecond dose, centrifuged, and plasma was analyzed by LiquidChromatography-Mass Spectrometry.

1(B) RAD1901 promoted survival in a mouse xenograft model of brainmetastasis (MCF-7 intracranial models).

The potential ability of RAD1901 to cross the blood-brain barrier andinhibit tumor growth was further evaluated using an MCF-7 intracranialtumor xenograft model.

Female athymic nude mice (Crl:NU(NCr)-Foxnlmu) were used for tumorxenograft studies. Three days prior to tumor cell implantation, estrogenpellets (0.36 mg E2, 60-day release, Innovative Research of America,Sarasota, Fla.) were implanted subcutaneously between the scapulae ofall test animals using a sterilized trochar. MCF-7 human breastadenocarcinoma cells were cultured to mid-log phase in RPMI-1640 mediumcontaining 10% fetal bovine serum, 100 units/mL penicillin G, 100 μg/mLstreptomycin sulfate, 2 mM glutamine, 10 mM HEPES, 0.075% sodiumbicarbonate and 25 g/mL gentamicin. On the day of tumor cell implant,the cells were trypsinized, pelleted, and resuspended in phosphatebuffered saline at a concentration of 5×10′ cells/mL. Each test mousereceived 1×10⁶ MCF-7 cells implanted intracranially.

Five days after tumor cell implantation (designated as day 1 of thestudy), mice were randomized into three groups of 12 animals each andtreated with vehicle, fulvestrant (0.5 mg/animal daily), or RAD1901 (120mg/kg daily), as described above.

The endpoint was defined as a mortality or 3× survival of the controlgroup, whichever comes first. Treatment tolerability was assessed bybody weight measurements and frequent observation for clinical signs oftreatment-related adverse effects. Animals with weight loss exceeding30% for one measurement, or exceeding 25% for three measurements, werehumanely euthanized and classified as a treatment-related death.Acceptable toxicity was defined as a group-mean body weight loss of lessthan 20% during the study and not more than one treatment-related deathamong ten treated animals, or 10%. At the end of study animals wereeuthanized by terminal cardiac puncture under isoflurane anesthesia.RAD1901 and fulvestrant concentration in plasma and tumor weredetermined using LC-MS/MS.

Kaplan Meier survival analysis demonstrated that RAD1901 significantlyprolonged survival compared to fulvestrant (P<0.0001; FIG. 8). Noanimals in the control or fulvestrant group survived beyond day 20 andday 34 respectively, whereas 41% (5/12) of the RAD1901 treated animalssurvived until the end of the study on day 54.

Concentration of RAD1901 in the plasma was 738±471 ng/mL and in theintracranial tumor was 462±105 ng/g supporting the hypothesis thatRAD1901 is able to effectively cross the blood-brain barrier. Incontrast, concentrations of fulvestrant were substantially lower in theplasma (21±10 ng/mL) and in the intracranial tumor (8.3±0.8 ng/g).

1(C). Phase 1 study of RAD1901 treatment for ER+ advanced breast cancer.

In the phase 1 study, safety, tolerability and pharmacokinetics wereevaluated in 44 healthy postmenopausal females. No dose limitingtoxicities were observed, maximum tolerated dose (MTD) was notestablished. Plasma exposure increased more than dose proportionallyover the dose range tested.

Subjects

8 postmenopausal females with advanced adenocarcinoma of the breast (ER+tumor with no less than 1% staining by IHC, HER2-negative tumor withECOG performance status of 0 or 1) were enrolled as subjects for thisphase 1 study. The subjects must have received the following priortreatments:

-   -   no greater than 2 prior chemotherapy regimens in the        advanced/metastatic setting    -   6 months prior endocrine therapy and had progressed on prior        endocrine therapy    -   Subjects with untreated or symptomatic CNS metastases or prior        anticancer treatment within the following windows were excluded:    -   Tamoxifen<14 days before _first dose study treatment    -   Fulvestrant<90 days before _first dose study treatment    -   Chemotherapy<28 days before _first dose study treatment    -   LHRH analogue<12 months before _first dose study treatment

DLT Criteria

-   -   Any Grade no less than 3 non-hematologic toxicity (excluding        alopecia and nausea, vomiting or diarrhea that has not been        treated with optimal medication)    -   Any Grade no less than 3 hematologic toxicity    -   Any grade toxicity that leads to study drug interruption for >7        days    -   Dose limiting toxicity observation period is day 1-28 of Cycle 1

Treatment Emergent Adverse Events (TEAEs)

TEAEs were recorded throughout the study. Preliminary data aresummarized in Table 12. “n” is number of subjects with at least onetreatment-related AE in a given category, AEs graded as per the CommonTerminology Criteria for Adverse Events (CTCAE) v4.0, and any patientwith multiple scenarios of a same preferred term was counted only onceto the most severe grade. No death or dose limiting toxicities wereobserved, maximum tolerated dose (MTD) was not established. Most AEswere grade 1 or 2. Most common treatment-related AEs were dyspepsia (⅝patients) and nausea (⅜ patients). Two serious AEs (SAES) were observed,one a grade 3 treatment-related constipation, and the other shortness ofbreath (pleural effusion) not related to the treatment.

The heavily pretreated subjects of this phase 1 study included subjectspreviously treated with multiple endocrine and targeted agents, e.g.,CDK4/6, PI3K and mTOR inhibitors. No dose limiting toxicities wereobserved after RAD1901 treatment at 200 mg daily oral dose up to 6months, and at 400 mg daily oral dose up to two months. Thus, RAD1901showed potential for treating ER+advanced breast cancer, especially insubjects previously treated with endocrine and/or targeted agents suchas CDK4/6, PI3K and mTOR inhibitors.

Example II RAD1901 Preferably Accumulated in Tumor and Could beDelivered to Brain

MCF-7 xenografts as described in Example I(A)(i) were further evaluatedfor RAD1901 concentration in plasma and tumor using LC-MS/MS. At the endof study, the concentration of RAD1901 in plasma was 344±117 ng/mL andin tumor in 11,118±3,801 ng/mL for the 60 mg/kg dose level. A similartumor to plasma ratio was also observed at lower dose levels where tumorconcentrations were approximately 20-30 fold higher than in plasma.RAD1901 levels in plasma, tumor, and brain for mice treated for 40 daysare summarized in Table 1. A significant amount of RAD1901 was deliveredto the brain of the treated mice (e.g., see the B/P ratio (RAD1901concentration in brain/the RAD1901 concentration in plasma)), indicatingthat RAD1901 was able to cross the blood-brain barrier (BBB).Unexpectedly, RAD1901 preferably accumulated in the tumor. See, e.g.,the T/P (RAD1901 concentration in tumor/RAD1901 concentration in plasma)ratio shown in Table 1.

Example III RAD1901 Inhibited ER Pathway and Degraded ER

III(A). RAD1901 decreased ER-engagements in uterus and pituitary inhealthy postmenopausal female human subjects.

The subjects had an amenorrhea duration of at least 12 months and serumFSH consistent with menopause. The subjects were 40-75 years old withBMI of 18.0-30 kg/m². Subjects had intact uterus. Subjects havingevidence of clinically relevant pathology, increased risk of stroke orof history venous thromboembolic events, or use of concomitantmedication less than 14 days prior to admission to clinical researchcenter (paracetamol allowed up to 3 days prior) were excluded.

FES-PET was performed at baseline and after 6 days of exposure toRAD1901 to evaluate ER engagement in the uterus. RAD1901 occupied 83%and 92% of ER in the uterus at the 200 mg (7 subjects) and 500 mg (6subjects) dose levels, respectively.

FES-PET imaging showed significant reduction in binding oflabelled-estradiol to both the uterus and pituitary after RAD1901treatment with 200 mg or 500 mg (once/day, p.o., 6 days).

Due to the high ER expression, the uterus showed a strong FES-PET signalat baseline before RAD1901 treatment (FIG. 9A, baseline transversal viewfor uterus FES-PET scan of Subject 3 treated with 200 mg dose level;FIG. 9B, baseline sagittal view and transversal view for uterus FES-PETscan respectively of Subject 7 treated with 500 mg dose level). However,when scanned four hours post dosing on day 6 in the study, the uteruswas hardly visible (at or close to background FES-PET signal (FIG. 9A,Day 6 transversal view for uterus scan of Subject 3; and FIG. 9B, Day 6sagittal view and transversal view for uterus scan respectively ofSubject 7). Such data were consistent with ER degradation and/orcompetition for the binding to the receptor. FIGS. 9A and 9B alsoinclude CT scan of the uterus scanned by FES-PET showing the existenceof the uterus before and after RAD1901 treatment.

The FES-PET uterus scan results were further quantified to show thechange of post-dose ER-binding from baseline for 7 subjects (FIG. 9C),showing Subjects 1-3 and Subjects 4-7 as examples of the 200 mg dosegroup and 500 mg dose group, respectively. RAD1901 showed robust ERengagement at the lower dose level (200 mg).

FIGS. 10A-B showed a representative image of FES-PET scan of the uterus(A) and pituitary (B) before (Baseline) and after (Post-treatment)RAD1901 treatment at 500 mg p.o. once a day, after six days. FIG. 10Ashowed the FES-PET scan of the uterus by (a) Lateral cross-section; (b)longitude cross-section; and (c) longitude cross-section.

The subject's post dose FES-PET scan of uterus and pituitary showed nonoticeable signal of ER binding at uterus (FIG. 10A, Post-treatment) andat pituitary (FIG. 10B, Post-treatment), respectively.

Thus, the results showed that RAD1901 effectively degraded/deactivatedER in human at a dosage of 200 and 500 mg PO once/day, after six days.

Standard uptake value (SUV) for uterus, muscle and bone were calculatedand summarized for RAD1901 treatments at 200 mg and 500 mg p.o. daily inTables 2 and 3, respectively. Post-dose uterine signals were a tor closeto levels from “non-target tissues,” suggesting a complete attenuationof FES-PET uptake post RAD1901 treatment. Almost no change was observedin pre-versus post-treatment PET scans in tissues that did notsignificant express estrogen receptor.

Thus, RAD1901 or salt or solvate (e.g., hydrate) thereof may be used intreating cancer and/or tumor cells having overexpression of ER (e.g.,breast cancer, uterus cancer, and ovary cancer), without negativeeffects to other organs (e.g. bones, muscles). RAD1901 or salt orsolvate (e.g., hydrate) thereof may be especially useful in treatingmetastatic cancers and/or tumors having overexpression of ER in otherorgans, e.g., the original breast cancer, uterus cancer, and/or ovarycancer migrated to other organs (e.g., bones, muscles), to treat breastcancer, uterus cancer, and/or ovary cancer lesions in other organs(e.g., bones, muscles), without negative effect to said organs.

III(B). RAD1901 decreased ER expression and inhibited ER pathway.

III(B)(i)(1) RAD1901-everolimus combo was more effective in decreasingER and PR expression in MCF-7 xenograft models and treatment withRAD1901, everolimus or fulvestrant alone, or a ful-everolimuscombination.

Treatment with the combination of RAD1901 and everolimus was also moreeffective at decreasing ER and PR expression in vivo in the MCF-7xenograft models (as described in Example I(A)(ii)) than treatment withRAD1901, everolimus, or fulvestrant alone, or treatment with acombination of fulvestrant and everolimus (FIG. 11); tumors harvestedtwo hours after the last dosing).

III(B)(i)(2) Comparison of RAD1901 and fulvestrant in MCF-7 and T47Dcell lines.

The effects of RAD1901 and fulvestrant were compared using MCF-7 andT47D cell lines, both are human breast cancer cell lines, at variousconcentrations, 0.01 μM, 0.1 μM and 1 M (FIG. 12A for MCF-7 cell lineassays; and FIG. 12B for T47D cell lines). Three ER target genes,progesterone receptor (PgR), growth regulation by estrogen in breastcancer 1 (GREB1) and trefoil factor 1 (TFF 1), were used as markers.RAD1901 caused nearly complete ER degradation and inhibited ER signaling(FIGS. 12A-B). Especially for MCF-7 cell lines, fulvestrant showedcomparable or even slightly higher efficacies in inhibiting ER signalingwhen administered at the same concentration. Unexpectedly, RAD1901 wascomparable or more effective than fulvestrant in inhibiting tumorgrowth, and driving tumor regression as disclosed supra in Example I(A)and Example I(B).

III(B) (1) (3) RAD1901 treatment resulted in ER degradation andabrogation of ER signaling in MCF-7 Xenograft Model-described supra inExample 1(A)(ii)(1).

RAD1901 treatment resulted in ER degradation in vivo (FIGS. 13A and 13B,student's t-test: *p-value<0.05, **p-value<0.01) and inhibited of ERsignaling in vivo (FIGS. 13A and 13C, student's t-test: *p-value<0.05,**p-value<0.01).

Tumor harvested from MCF-7 xenograft 2 hours after the final dose ofRAD1901 (30 mg/kg, 60 mg/kg, p.o., q.d.) or fulvestrant (3 mg/dose,s.c., qwk) showed significantly decreased ER and PR expression (FIGS.13A-B). Tumor harvested from MCF-7 xenograft 8 hours after the finaldose of fulvestrant treatment showed increased PR and ER expression.However, tumor harvested from MCF-7 xenograft 8 hours after the finaldose of RAD1901 treatment showed reduced PR and ER expression (FIGS. 13Aand 13C).

Tumor harvested from MCF-7 xenograft 8 hours or 12 hours after thesingle dose of RAD1901 (30 mg/kg, 60 mg/kg, or 90 mg/kg, p.o., q.d.)showed rapidly decreased PR expression (FIGS. 14A-C). Tumor harvestedfrom MCF-7 xenograft 4 hours or 24 hours after the 7th dose of RAD1901(30 mg/kg, 60 mg/kg, or 90 mg/kg, p.o., q.d.) showed consistent andstable inhibition of ER signaling (FIG. 14B). Quantification of westernblot analyses of tumor harvested from MCF-7 xenograft at various timepoints during the treatment of RAD1901 (30 mg/kg, 60 mg/kg, or 90 mg/kg,p.o., q.d.) showed a dose-dependent decrease in PR (FIG. 14C).

RAD1901 treatment caused a rapid decrease in proliferation in MCF-7xenograft models. For example, tumor harvested from MCF-7 xenograftmodels 8 hours after the single dose of RAD1901 (90 mg/kg, p.o., q.d.)and 24 hours after the 4th dose of RAD1901 (90 mg/kg, p.o., q.d.) weresectioned and stained to show a rapid decrease of the proliferationmarker Ki67 (FIGS. 15A and 15B).

These results suggest that RAD1901 treatment results in ER degradationand inhibition of ER signaling in ER WT xenografts in vivo.

III(B)(i)(4) RAD1901 treatment resulted in ER degradation and abrogationof ER signaling in PDx-4 models described supra in Example 1 (A)(ii).

RAD1901 treatment caused a rapid decrease in proliferation in the PDx-4models. For example, four hours after the final dose on the last day ofa 56 day efficacy study, tumor harvested from PDx-4 models treated withRAD1901 (30, 60, or 120 mg/kg, p.o., q.d.) or fulvestrant (1 mg/animal,qwk) were sectioned and showed a rapid decrease of the proliferationmarker Ki67 compared to PDx-4 models treated with fulvestrant (FIG. 16).

These results suggest that RAD1901 treatment results in ER degradationand inhibition of ER signaling in ER WT xenografts in vivo.

III(B)(ii) RAD1901 treatment resulted in decreased ER signaling in amutant ER PDx-5 models described supra in Example I(A)(iii)(1).

Tumors were harvested at the indicated time points after the last day ofdosing (unless otherwise specified), homogenized in RIPA buffer withprotease and phosphatase inhibitors using a Tissuelyser (Qiagen). Equalamounts of protein were separated by MW, transferred to nitrocellulosemembranes and blotted with the following antibody as described in theMaterials and methods section: progesterone receptor (PR, Cell SignalingTechnologies; 3153).

Bands were quantified using 1D Quant software (GE), and PR IHC Allredscores obtained from PDx-5 models as described in Example I(A)(iii)(1)are shown in FIG. 17. Fulvestrant exerted little influence to PRexpression, while RAD1901 showed efficacy at dosages of both 60 mg/kgand 120 mg/kg (q.d., p.o., FIG. 17).

These results indicate that for tumors expressing certain ERα mutations(e.g., Y537S), RAD1901 was more effective than fulvestrant at inhibitingthe tumor growth, especially effective in inhibiting the growth oftumors which were hardly responsive to fulvestrant treatment (e.g., at adosage of 3 mg/dose, qwk, s.c., FIG. 6A PDx-5). Furthermore, for thetumors which did not respond well to fulvestrant treatment (e.g.,PDx-5), RAD1901 was effective in reducing PR expression in vivo, whilefulvestrant was not (FIG. 17).

Example IV Impact of RAD1901 Treatment to Uterine Tissue and/or BMD

IV(A(1)): RAD1901 antagonized estradiol stimulation of uterine tissue.

The uterotropic effects of RAD1901 were investigated by assessingchanges in uterine weight, histology, and C3 gene expression in immaturerats. Results from a representative study are shown in FIGS. 18A-D.

Assessment of Uterotropic Activity

Sprague-Dawley rat pups were weaned at 19 days of age, randomized intogroups (n=4), and administered vehicle (aqueous methylcellulose), E2(0.01 mg/kg), raloxifene (3 mg/kg), tamoxifen (1 mg/kg), RAD1901 alone(0.3 to 100 mg/kg), or RAD1901 (0.01 to 10 mg/kg) in combination with E2(0.01 mg/kg), either by subcutaneous injection or by oral gavage asappropriate (see reagents, above) once daily for 3 consecutive days.Twenty-four hours after the final dose, all animals were euthanized bycarbon dioxide inhalation. Body weights and wet uterine weights wererecorded for each animal. Similar assays were also conducted withRAD1901 (0.03 to 100 mg/kg) in rats and mice (Charles RiverLaboratories, Montreal, QC).

Fresh uterine tissue from each rat was fixed in 4% paraformaldehyde,dehydrated with ethanol, and embedded into JB4 plastic resin. Sectionswere cut at 8 μm and stained with 0.1% Toluidine Blue O. Thickness ofthe endometrial epithelium was measured using a Zeiss Axioskop 40microscope using the Spot Advanced program; the mean of 9 measurementsper specimen was calculated.

Uterine Complement Component 3 (C3) Gene Expression

To determine relative expression levels of C3 in the treated uterinetissue, RNA was extracted from the remaining tissue using the Micro toMidi Total RNA Purification Kit (Invitrogen, Carlsbad, Calif.) accordingto the manufacturer's instructions. RNA was quantified, and equalamounts were reverse-transcribed using the High Capacity cDNA ArchiveKit (Applied Biosystems, Foster City, Calif.).

Quantitative PCR was performed using the ABI Prism 7300 System (AppliedBiosystems). PCR was done using the Taqman Universal Master Mix withprobe sets for C3 and for the 18S ribosomal RNA as a reference gene.Thermal cycling conditions comprised an initial denaturation step at 95°C. for 10 min, followed by 40 cycles at 95° C. for 15 second and 60° C.for 1 minute.

Relative gene expression was determined by normalizing each sample tothe endogenous control (18S) and comparing with a calibrator (vehicle).Relative gene expression was determined using the following equation:2-ΔΔCt (where Ct=cycle threshold or the cycle number at which PCRproduct was first detected, ΔCt=normalized sample value, andΔΔCt=normalized difference between dosed subjects and the vehicle). Fivereplicate gene expression determinations were conducted for each dose,within each study.

Treatment with E2 (0.01 mg/kg), raloxifene (RAL, 3 mg/kg) or tamoxifen(TAM, 1 mg/kg) resulted in significant increases in uterine wet weightcompared to vehicle alone, whereas RAD1901 treatment at a range of dosesbetween 0.3 and 100 mg/kg did not significantly affect uterine wetweight (FIG. 18A). Data shown (FIG. 18A) are means (±SEM); n=4 rats pergroup; P vs. vehicle: *<0.05; vs. E2‡<0.05. Further, when administeredin combination with E2 (0.01 mg/kg), RAD1901 antagonized E2-mediateduterine stimulation in a dose-dependent manner, exhibiting significantinhibition of uterotropic activity at doses of 0.1 mg/kg and greater andcomplete inhibition at 3 mg/kg. The EC₅₀ for RAD1901 was approximately0.3 mg/kg. Similar results were obtained in mice where RAD1901 doses0.03 to 100 mg/kg also had no effect on uterine wet weight or epithelialthickness (data not shown).

Treatment-dependent changes in uterine tissue were further investigatedby quantitative microscopic histology. There was a statisticallysignificant increase in endometrial epithelial thickness after treatmentwith E2 at both 0.01 and 0.3 mg/kg (FIG. 18B). A significant increase inepithelial thickness was also observed after treatment with tamoxifen (1mg/kg) or raloxifene (3 mg/kg). In contrast, RAD1901 treatment did notincrease endometrial epithelial thickness up to the highest evaluateddose of 100 mg/kg. Representative images of the endometrial epitheliumare shown in FIG. 18C.

Consistent with the changes in both uterine weight and endometrialepithelial thickness, E2, tamoxifen, and raloxifene all significantlyincreased the expression of the estrogen-regulated complement gene, C3(FIG. 18D). In contrast, RAD1901 did not increase C3 gene expression atany of the doses tested (0.3 to 100 mg/kg). Furthermore, RAD1901 at 1, 3and 10 mg/kg significantly suppressed E2-stimulated C3 gene expression.

RAD1901 Did Not Stimulate the Uterus of Immature Female Rats

Immature female rats were administered (orally) once daily, for 3consecutive days with vehicle (VEH), estradiol (E2), Raloxifene (RAL),Tamoxifen (TAM), RAD1901 or RAD1901+E2. Wet uterine weights weremeasured. Data shown (FIGS. 18A-D) are means (±SEM); n=4 rats per group;P vs. vehicle: *<0.05; vs. E2‡<0.05.

Example II(A)(2) Treatment with RAD1901 Protected Against Bone Loss inOvariectomized Rats

The bone-specific effects of RAD1901 was examined in ovariectomizedrats.

As a model of postmenopausal bone loss, ovariectomy was performed onanesthetized adult female Sprague-Dawley rats, with sham surgery as acontrol. Following surgery, ovariectomized rats were treated once dailyfor 4 weeks with vehicle, E2 (0.01 mg/kg), or RAD1901 (0.1, 0.3, 1, 3mg/kg), administered as described above, with 20 animals per group.Animals in the sham surgery group were vehicle treated. All animals wereeuthanized by carbon dioxide inhalation 24 hours after the final dose.Bone mineral density was assessed at baseline and again after 4 weeks oftreatment using PIXImus dual emission x-ray absorptiometry.

At necropsy, the left femur of each animal was removed, dissected freeof soft tissue and stored in 70% ethanol before analysis. A detailedqualitative and quantitative 3-D evaluation was performed using amicro-CT40 system (Scanco Systems, Wayne, Pa.). For each specimen, 250image slices of the distal femur metaphysis were acquired. Morphometricparameters were determined using a direct 3-D approach in pre-selectedanalysis regions. Parameters determined in the trabecular bone includedbone volume density, bone surface density, trabecular number, trabecularthickness, trabecular spacing, connectivity density, and apparent bonedensity.

Following ovariectomy, untreated (vehicle control) rats experienced adecrease in bone mineral density both in the whole full femur and in thelumbar spine compared to baseline (Table 4). Treatment with E2 wasassociated with prevention of bone loss in both the femur and spine.Treatment with RAD1901 resulted in a dose-dependent and statisticallysignificant suppression of ovariectomy-induced bone loss (data shown forthe 3 mg/kg treatment group). At doses of 0.1 mg/kg to 3 mg/kg, bonemineral density in RAD1901-treated rats was complete, with nostatistically significant difference from the E2-treated group.

Micro-CT analysis of the distal femur (Table 5) demonstrated thatovariectomy induced significant changes in a number of keymicro-architectural parameters when compared to sham surgery animals.These changes were consistent with a decrease in bone mass and includedecreased bone volume, reduced trabecular number, thickness and density,and increased trabecular separation. Consistent with the preservation ofbone mineral density observed after treatment with RAD1901, significantpreservation of trabecular architecture was observed in keymicro-structural parameters (Table 5)

Example IV(B) Phase 1 Dose Escalation Study of RAD101 in HealthyPostmenopausal Women

In the phase 1 study, safety, tolerability and pharmacokinetics wereevaluated in 44 healthy postmenopausal females. No dose limitingtoxicites (DLT) were observed, maximum tolerated dose (MTD) was notestablished. Plasma exposure increased more than dose proportionallyover the dose range tested.

Subjects

44 healthy postmenopausal females were enrolled as subjects for thisphase 1 study. The subjects had an amenorrhea duration of at least 12months and serum FSH consistent with menopause. The subjects were 40-75years old with BMI of 18.0-30 kg/m². Subjects having evidence ofclinically relevant pathology, increased risk of stroke or of historyvenous thromboembolic events, or use of concomitant medication less than14 days prior to admission to clinical research center (paracetamolallowed up to 3 days prior) were excluded.

Dosing

The subjects were treated with placebo or at least one oral dose dailyafter a light breakfast for 7 days at dose levels of 200 mg, 500 mg, 750mg and 1000 mg, respectively. The key baseline demographics of the 44healthy postmenopausal females enrolled in the phase 1 study aresummarized in Table 6.

Treatment Emergent Adverse Events (TEAEs)

TEAEs were recorded, and the most frequent (>10% of patients in thetotal active group who had any related TEAEs) adverse events (AEs) aresummarized in Table 7, “n” is number of subjects with at least onetreatment-related AE in a given category, AEs graded as per the CommonTerminology Criteria for Adverse Events (CTCAE) v4.0, and any patientwith multiple scenarios of a same preferred term was counted only onceto the most severe grade. No dose limiting toxicites were observed,maximum tolerated dose (MTD) was not established.

Pharmacokinetic Evaluations

A series of blood samples were taken during the study for the analysisof RAD1901 in plasma. Blood samples of 5 mL each were taken via anindwelling IV catheter or by direct venipuncture into tubes containingK3-EDTA as anticoagulant. Steady state was achieved by day 5 oftreatment. Geometric Mean (Geo-Mean) plasma concentration-time profilesof RAD1901 were evaluated. Plasma pharmacokinetic results of the groupstreated with RAD1901 (200, 500, 750 or 1,000 mg) on Day 7 (N=35) in thestudy are provided in Table 8 and FIG. 19, as an example. The median tv2was between 37.5-42.3 hours (Table 8). After multiple doseadministration of RAD1901, median tmax ranged between 3-4 hourspost-dose.

Example V(A)-1 Modeling of RAD1901-ERα Binding Using Select Published ERStructures

Unless specified otherwise, when structures are shown by their stickmodel, each end of a bond is colored with the same color as the atom towhich it is attached, wherein grey is carbon, red is oxygen, blue isnitrogen and white is hydrogen.

Fourteen published structures (i.e., models) of ERα ligand-bindingdomain (LBD) complexed with various ER ligands were selected from 96published models by careful evaluation. One of these fourteen models was3ERT (human ERα LBD bound to 4-hydroxytamoxifen (OHT)). OHT is theactive metabolite of tamoxifen and a first generation SERM thatfunctions as an antagonist in breast tissue.

In 3ERT (FIGS. 20 and 21), the ERα binding site adopts a three layer“helical sandwich” forming a hydrophobic pocket which includes Helix 3(H3), Helix 5 (H5), and Helix 11 (H11) (FIG. 20). The dotted box in FIG.21 represents the binding site and residues within the binding site thatare important or are effected by OHT binding. OHT functions as anantagonist by displacing H12 into the site where LXXLL coactivator(s)binds. OHT occupies the space normally filled by L540 and modifies theconformation of four residues on the C-terminal of Helix 11 (G521, H524,L525, and M528). OHT also forms a salt bridge with D351, resulting incharge neutralization.

The other thirteen ERα LBD-ER ligand models were compared to 3ERT.Differences in their residue poses are summarized in Table 10.Superimposition of the ERα structures of the fourteen models (FIG. 22)shows that these structures differed significantly at residues E380,M421, G521, M522, H524, Y526, S527, M528, P535, Y537, L540, and variouscombinations thereof.

Root-mean-square deviation (RMSD) calculations of any pair of thefourteen models are summarized in Table 11. Structures were consideredto be overlapping when their RMSD was <2 Å. Table 11 shows that allfourteen models had a RMSD<1.5 Å. Using conditional formatting analysissuggested that 1R5K and 3UUC were the least similar to the other models(analysis not shown). Therefore, 1R5K and 3UUC were considered a unique,separate structural cluster to be examined.

ERα residues bound by ligand in the fourteen models are summarized inTable 12. Table 12 also shows the EC₅₀ in the ERα LBD-antagonistcomplexes. Out of the fourteen models, thirteen showed H-bondinteractions between the ligand and E353; twelve showed pi interactionsbetween the ligand and F404; five showed H-bond interactions between theligand and D351; six showed H-bond interactions between the ligand andH524; four showed H-bond interactions between the ligand and R394; andone (3UUC) showed interactions between the ligand and T347.

Each of the fourteen models was used to dock a random library of 1,000compounds plus the ligand the model was published with (the knownantagonist) to determine whether the model could identify and prioritizethe known antagonist. If the model could identify the known antagonist,the model was determined to be able to predict the pose of its ownpublished ligand. EF₅₀ was then calculated to quantify the model'sstrength to see how much better it was than a random selection. RAD1901was docked in the selected models (e.g., FIGS. 23A&B-27A&B). Dockingscores of the published ligand and RAD1901 in the models weredetermined. EC₅₀ was also determined. Visual inspection of RAD1901showed that it “obeyed” the interactions shown with the publishedligands in 1R5K, 1SJ0, 2JFA, 2BJ4, and 2OUZ. No spatial clashes werenoticed. In certain embodiments, e.g., in 1R5k and 2BJ4, RAD1901 had ahigher docking score than the published ligand.

The evaluation results of nine models (1ERR, 3ERT, 3UCC, 2IOK, 1R5K,1SJ0, 2JFA, 2BJ4, and 2OUZ) are summarized in Table 13.

1ERR and 3ERT could not predict the correct pose of their crystallizedligand. RAD1901 did not dock in 3UCC. The tetrahydronaphtaalen in2IOK-RAD1901 bound in a non-traditional manner.

The major differences between the models 1R5K, 1SJ0, 2JFA, 2BJ4, and2OUZ were the residues in the C-term of Helix 11 (G521-M528).

FIGS. 23A&B shows the modeling of RAD1901-1R5K (A) and GW5-1R5K (B).RAD1901 bound with H-bond interactions to E353, R394, and L536; and withp-interaction with F404.

FIG. 24A&B shows the modeling of RAD1901-1SJ0 (A) and E4D-1SJ0 (B).RAD1901 bound with H-bond interactions to E353, and D351; and withp-interaction with F404.

FIG. 25A&B shows the modeling of RAD1901-2JFA (A) and RAL-2JFA (B).RAD1901 bound with p-interaction with F404.

FIG. 26A&B shows the modeling of RAD1901-2BJ4 (A) and OHT-2BJ4 (B).RAD1901 bound with H-bond interactions with E353 and R394; andp-interaction with F404.

FIG. 27A&B shows the modeling of RAD1901-2IOK (A) and IOK-2IOK (B).RAD1901 bound with H-bond interactions with E353, R394, and D351; andp-interaction with F404.

The published ligands in the models have the following structures:

Example V(A)-2 Induced Fit Docking (IFD) of ERα with RAD1901 andFulvestrant

Binding conformation of RAD1901 in ERα was further optimized by IFDanalysis of the five ERα crystal structures 1R5K, 1SJO, 2JFA, 2BJ4, and2OUZ. IFD analysis accounted for the receptor flexibility (upon ligandbinding) to accommodate its correct binding conformation.

A library of different conformations for each ligand (e.g., RAD1901 andfulvestrant) was generated by looking for a local minima as a functionof rotations about rotatable bonds. The library for RAD1901 had 25different conformations.

The five ERα crystal structures were prepared and minimized. Thecorresponding ligand in the published X-ray structures were used todefine the ERα binding pocket.

RAD1901 conformations were docked into the prepared ERα structureswherein they were allowed to induce side-chain or back-bone movements toresidues located in the binding pocket. Those movements allowed ERα toalter its binding site so that it was more closely conformed to theshape and binding mode of the RAD1901 conformation. In some examples,small backbone relaxations in the receptor structure and significantside-chain conformation changes were allowed in the IFD analysis.

An empirical scoring function was used to approximate the ligand bindingfree energy to provide a docking score or Gscore. Gscore is also knownas GlideScore, which may be used interchangeably with docking score inthis example. The docking score was an estimate of the binding affinity.Therefore, the lower the value of the docking score, the “better” aligand bound to its receptor. A docking score of −13 to −14 correspondedto a very good binding interaction.

The RAD1901 conformations resulted from the IFD analysis with 1R5K,1SJ0, 2JFA, 2BJ4, and 2OUZ respectively were superimposed to show theirdifferences (FIGS. 28-30A&B, shown in stick model). All bonds in eachRAD1901 conformation were shown in the same color in FIGS. 28, 29 and30A.

The RAD1901 conformations resulted from the IFD analysis with1R5K (blue)and 2OUZ (yellow) had N-benzyl-N-ethylaniline group of RAD1901 on thefront (FIG. 28). The RAD1901 conformations resulted from the IFDanalysis with 2BJ4 (green) and 2JFA (pink) had N-benzyl-N-ethylanilinegroup of RAD1901 on the back (FIG. 29). The RAD1901 conformationsresulted from the IFD analysis with 2BJ4 (green), 2JFA (pink) and 1SJ0(brown) were quite similar as shown by their superimpositions (FIGS. 30Aand 30B). The RAD1901 IFD docking scores are summarized in Table 14.

The IFD of RAD1901 with 2BJ4 showed hydrogen bond interactions with E353and D351 and pi-interactions with F404 (FIGS. 31A-31C). FIG. 31A showedregions within the binding site suitable for H-bond acceptor group(red), H-bond donor group (blue) and hydrophobic group (yellow). InFIGS. 31A and 31B, light blue was for carbon for RAD1901. FIGS. 32A-32Cshow a protein-surface interactions of the IFD of RAD1901 with 2BJ4.FIGS. 32A and 32B are the front view, and FIG. 32C is the side view. Themolecular surface of RAD1901 was blue in FIG. 32A, and green in FIG.32C. FIGS. 32B and 32C are electrostatic representation of the solventaccessible surface of ERα, wherein red represented electronegative andblue represented electropositive.

Similar IFD analysis was carried out for fulvestrant with 2BJ4 asdescribed supra. The fulvestrant-2BJ4 IFD resulted in a Gscore of−14.945 and showed hydrogen bond interactions with E353, Y526, and H524and pi-interactions with F404 (FIGS. 33A-33C). FIG. 33A showed regionswithin the binding site suitable for H-bond acceptor group (red), H-bonddonor group (blue) and hydrophobic group (yellow). In FIG. 33A, lightblue was for carbon for RAD1901.

FIGS. 34A and 34B showed RAD1901 and fulvestrant docked in 2BJ4 by IFDboth had pi-interactions with F404 and hydrogen bond interactions withE353. Furthermore, RAD1901 had hydrogen bond interaction with D351 (bluerepresenting RAD1901 molecular surface, FIG. 34B), while fulvestrant hadhydrogen bond interactions with Y526, and H524 (green representingfulvestrant molecular surface, FIG. 34C). Superimpositions of 2BJ4docked with RAD1901 and fulvestrant are shown in FIGS. 35A and 35B. InFIG. 35A, green represents fulvestrant molecular surface and bluerepresents RAD1901 molecular surface. In FIG. 35B, the brown structureis fulvestrant and the blue structure is RAD1901.

Example V(A)-3 Modeling Evaluation of Select ERα Mutations

Effects of various ERα mutations on the C-terminal ligand-binding domainwere evaluated. Specific ERα mutations evaluated were Y537X mutantwherein X was S, N, or C; D538G; and S463P.

Y537 resides in Helix 12. It may regulate ligand binding,homodimerization, and DNA binding once it is phosphorylated, and mayallow ERα to escape phosphorylation-mediated controls and provide a cellwith a potential selective tumorigenic advantage. In addition, it maycause conformational changes that makes the receptor constitutivelyactive.

The Y537S mutation favors the transcriptionally active closed pocketconformation, whether occupied by ligand or not. The closed butunoccupied pocket may account for ERα's constitutive activity (Carlsonet al. Biochemistry 36:14897-14905 (1997)). Ser537 establishes ahydrogen-bonding interaction with Asp351 resulting in an alteredconformation of the helix 11-12 loop and burial of Leu536 in asolvent-inaccessible position. This may contribute to constitutiveactivity of the Y537S mutant protein. The Y537S surface mutation has noimpact on the structure of the LBD pocket.

Y537N is common in ERα-negative metastatic breast cancer. A mutation atthis site may allow ERα to escape phosphorylation-mediated controls andprovide a cell with a potential selective tumorigenic advantage.Specifically, Y537N substitution induces conformational changes in theERα that might mimic hormone binding, not affecting the ability of thereceptor to dimerize, but conferring a constitutive transactivationfunction to the receptor (Zhang et al. Cancer Res 57:1244-1249 (1997)).

Y537C has a similar effect to Y537N.

D538G may shift the entire energy landscape by stabilizing both theactive and inactive conformations, although more preferably the active.This may lead to constitutive activity of this mutant in the absence ofhormones as observed in hormone-resistant breast cancer (Huang et al.,“A newfound cancer-activating mutation reshapes the energy landscape ofestrogen-binding domain,” J. Chem. Theory Comput. 10:2897-2900 (2014)).

None of these mutations are expected to impact the ligand binding domainnor specifically hinder RAD1901 binding. Y537 and D538 may causeconformational changes that leads to constitutive receptor activationindependent of ligand binding.

Example V(B) In vitro Binding Assay of ERα Constructs of Wildtype andLBD Mutant with RAD1901 and Other Compounds

In vitro binding assay of ERα constructs of wildtype (WT) and LBD mutantwith RAD1901 showed that RAD1901 bound to mutant ERα with a similaraffinity as to WT ERα.

ERα constructs of WT and LBD mutant were prepared by expressing andpurifying the corresponding LBD residues 302-552 with N-terminalthioredoxin and 6×His tags which were cleaved by TEV protease.

Fluorescence polarization (FP) was used to determine binding of testcompounds (RAD1901, fulvestrant, bazedoxifene, raloxifene, tamoxifene,and AZD9496) to ERα as per manufacturer's instructions (Polar Screen,Invitrogen) with 2 nM fluoromone, 100 nM ERα construct of WT or LBDmutant. Each set was carried out in duplicate and tested one testcompound to determine the IC50 for different ERα constructs (FIG. 36 forRAD1901 binding essay).

As stated above, the foregoing is merely intended to illustrate variousembodiments of the present invention. The specific modificationsdiscussed above are not to be construed as limitations on the scope ofthe invention. It will be apparent to one skilled in the art thatvarious equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein. All referencescited herein are incorporated by reference as if fully set forth herein.

TABLE 1 RAD1901 levels in plasma, tumor and brain of mice implanted withMCF7 cells after treated for 40 days. Dose Plasma Tumor Brain B/P T/P(mg/kg) (ng/mL) (ng/mL) (ng/mL) Ratio Ratio Vehicle BLQ* BLQ BLQ — —RAD1901 0.3 2 11 BLQ — RAD1901 1 3 45 BLQ — RAD1901 3 9 169 7 0.78 18.78RAD1901 10 39 757 14 0.36 19.41 RAD1901 30 137 3875 72 0.53 28.28RAD1901 60 334 11117 201 0.60 33.28 *BLQ: below the limit ofquantitation

TABLE 2 SUV for uterus, muscle, and bone for a human subject treatedwith 200 mg dose PO once/day for six days Uterus SUV Bone SUV Muscle SUVDose % Change % Change % Change 200 mg −85% 16% 0%

TABLE 3 SUV for uterus, muscle, and bone for human subjects (n = 4)treated with 500 mg dose PO once/day for six days. Uterus Muscle BoneMean SUV Mean SUV Mean SUV Subject Uterus Change Muscle Change BoneChange # Scan SUV (%) SUV (%) SUV (%) 1 Baseline 3.88 0.33 0.36 Day 60.58 −85 0.31 −6 0.48 33 2 Baseline 6.47 0.25 0.49 Day 6 0.33 −86 0.4268 0.55 12 3 Baseline 3.66 0.50 0.41 Day 6 0.58 −84 0.31 −38 0.47 −23 4Baseline 3.35 0.30 0.40 Day 6 0.41 −88 0.24 −20 0.52 30 Mean −86 1 13

TABLE 4 Effect of RAD1901 on BMD in ovariectomized rats.^(a) Femur BMDLumbar Spine BMD Treatment (% change) (% change) Sham 3.1 ± 2.4* 2.7 ±5.0* OVX + veh −5.4 ± 5.1 −10.2 ± 12.8 OVX + E2 −0.5 ± 2.6* −2.1 ± 12.2*OVX + RAD1901 0.4 ± 2.8* −1.1 ± 7.9* ^(a)Adult female rats underwenteither sham or ovariectomy surgery before treatment initiation withvehicle, E2 (0.01 mg/kg) or RAD1901 (3 mg/kg) once daily (n = 20 pertreatment group). BMD was measured by dual emission x-ray absorptiometryat baseline and after 4 weeks of treatment. Data are expressed as mean ±SD. *P < 0.05 versus the corresponding OVX + Veh control. BMD, bonemineral density; E2, beta estradiol; OVX, ovariectomized; Veh, vehicle.

TABLE 5 Effect of RAD1901 on femur microarchitecture in ovariectomizedrats^(a) BV/TV ConnD TbN TbTh TbSp ABD Treatment (%) (1/mm³) (1/mm) (mm)(mm) (mgHA/ccm) Sham 0.394 ± 0.069* 138 ± 21* 5.2 ± 0.6* 0.095 ± 0.008*0.175 ± 0.029* 456 ± 61* OVX + Veh 0.234 ± 0.065  91 ± 32 3.5 ± 0.90.085 ± 0.011 0.307 ± 0.086 301 ± 69 OVX + E2 0.309 ± 0.079* 125 ± 25*4.8 ± 0.8* 0.086 ± 0.008 0.204 ± 0.054* 379 ± 75* OVX + RAD1901 0.300 ±0.066* 113 ± 22* 4.5 ± 0.8* 0.088 ± 0.008 0.218 ± 0.057* 370 ± 66*^(a)Adult female rats underwent either sham or ovariectomy surgerybefore treatment initiation with vehicle, E2 (0.01 mg/kg) or RAD1901 (3mg/kg) once daily (n = 20 per treatment group). After 4 weeks, Bonemicroarchitecture was evaluated using microcomputed tomography. Data areexpressed as mean ± SD. *P < 0.05 versus the corresponding OVX + Vehcontrol. ABD, apparent bone density; BV/TV, bone volume density; ConnD,connectivity density; E2, beta estradiol; OVX, ovariectomized; TbN,trabecular number; TbTh, trabecular thickness; TbSp, trabecular spacing;Veh, vehicle.

TABLE 6 Key baseline demographics of Phase 1 dose escalation study ofRAD1901 RAD1901 RAD1901 RAD1901 RAD1901 Placebo 200 mg 500 mg 750 mg1,000 mg (N = 8) (N = 15) (N = 14) (N = 8) (N = 7) Race white 8(100)14(93) 10(71) 8(100) 7(100) (% of the cohort) Mean age, 64 62 59 64 64years Mean BMI, 26.1 25 24.4 24.9 26.7 kg/m²

TABLE 7 Most frequent (>10%) treatment related AEs in a Phase 1 doseescalation study of RAD1901 Total Placebo 200 mg 500 mg 750 mg 1000 mgTotal Active TEAE N = 8 N = 15 N = 14 N = 8 N = 7 N = 44 N = n(%) n(%)n(%) n(%) n(%) n(%) 44 Gr1 Gr2 Gr3 Gr1 Gr2 Gr3 Gr1 Gr2 Gr3 Gr1 Gr2 Gr3Gr1 Gr2 Gr3 Gr1 Gr2 Gr3 All Nausea 2 0 0 5 0 0 3 2 0 2 1 0 4 2 0 14 5 019 (25) (33) (21) (14) (25) (13) (57) (29) (32) (11) (43) Dyspepsia 1 00 3 0 0 5 2 0 4 0 0 1 1 0 13 3 0 16 (13) (20) (36) (14) (50) (14) (14)(30) (7) (36) Vomiting 0 0 0 2 0 0 1 5 1 0 2 0 0 3 0 3 10 1 14 (13) (7)(36) (7) (25) (43) (7) (23) (2) (32) Hot flush 1 0 0 2 0 0 6 0 0 2 0 0 10 0 11 0 0 11 (13) (13) (43) (25) (14) (25) (25) Abdominal 1 0 0 2 2 0 30 0 1 0 0 1 1 0 7 3 0 10 pain (13) (13) (13) (21) (13) (14) (14) (16)(7) (23) Oesophageal 0 0 0 0 2 0 1 3 0 1 0 0 1 1 1 3 6 1 10 pain (13)(7) (21) (13) (14) (14) (14) (7) (14) (2) (23) Headache 0 0 0 3 0 0 1 10 3 0 0 2 0 0 9 1 0 10 (20) (7) (7) (38) (29) (20) (2) (23) Hiccups 0 00 1 0 0 4 0 0 2 0 0 2 0 0 9 0 0 9 (7) (29) (25) (29) (20) (20) Salivary0 0 0 2 0 0 2 0 0 2 0 0 2 0 0 8 0 0 8 hyper- (13) (14) (25) (29) (18)(18) secretion Diarrhoea 1 0 0 0 0 0 3 0 0 0 0 0 3 1 0 6 1 0 7 (13) (21)(43) (14) (14) (2) (16) Dysphagia 0 0 0 0 0 0 1 2 0 3 1 0 0 0 0 4 3 0 7(7) (14) (38) (13) (9) (7) (16) Sensation 0 0 0 2 0 0 1 0 0 0 0 0 4 0 07 0 0 7 of a (13) (7) (57) (16) (16) foreign body Abdominal 0 0 0 1 1 01 0 0 1 0 0 2 0 0 5 1 0 6 distension (7) (7) (7) (13) (29) (11) (2) (14)Odynophagia 0 0 0 2 0 0 1 1 0 0 0 0 1 1 0 4 2 0 6 (13) (7) (7) (14) (14)(9) (5) (14) Dizziness 2 0 0 1 0 0 2 0 0 1 0 0 1 0 0 5 0 0 5 (25) (7)(14) (13) (14) (11) (11) Abdominal 0 0 0 3 0 0 0 0 0 1 1 0 0 0 0 4 1 0 5discomfort (20) (13) (13) (9) (2) (11) Flatulance 0 0 0 2 0 0 2 0 0 1 00 0 0 0 5 0 0 5 (13) (14) (13) (11) (11) Myalgia 1 0 0 2 1 0 0 1 0 1 0 00 0 0 3 2 0 5 (13) (13) (7) (7) (13) (7) (5) (11)

TABLE 8 Pharmacokinetic parameters in a Phase 1 dose escalation study ofRAD1901 (Day 7) 200 mg 500 mg 750 mg 1000 mg Parameter Statistic N = 15N = 11 N = 6 N = 3 C_(max) Geo-Mean 49.8 197 322 540 (ng/mL) Min, Max30.6, 85.5 105, 316 248, 420 481, 602 t_(max) (h) Median 3.00 4.00 3.004.00 Min, Max 2.00, 6.00 2.00-6.02 3.00, 4.00 3.00, 6.00 AUC_(0-tau)Geo-Mean 670 2927 4614 8292 (h*ng/mL) Min, Max 418, 1181 1562, 54603209, 7183 7281, 8947 t_(1/2) (h) Geo-Mean 38.3 37.5 38.4 42.3 Min, Max27.7, 51.4 33.8, 41.3 34.6, 46.4 38.7, 49.4

TABLE 9 Frequency of LBD mutations Frequency (%) D538G 29.5 Y537S 25.0Y537N 13.6 Y537C 9.1 E380Q 6.8 S463P 4.5 L536R 2.3 L536Q 2.3 P535H 2.3V392I 2.3 V534E 2.3

TABLE 10 Differences of ER-α LBD-antagonist complexes in residue posesversus 3ERT L1-3/ Helix Heli Residue 8 Helix 11 x 5 Helix 12 #/ M42 I42E52 M52 H52 L52 Y52 S52 M52 E38 Y53 L54 PDB 1 4 1 2 4 5 6 7 8 0 7 0 2BJ4x x x x x x x NA 2JFA x x x x x x x NA 1SJ0 x x x x x x x x 2JF9 x x x xx x x NA 1YIM x x x x x x 1R5K x x x x x x X x x IUM x x x x O 1ERR x xx x x x 2IOK x x x x x x x x 3UUC x x x x x x x x 1YIN x x x X x x x x x2AYR x X x x 2OUZ x x x x

TABLE 11 Evaluation of structure overlap of ER-α LBD-antagonistcomplexes by RMSD calculations: RMSD 3ERT 2BJ4 2JFA 1SJ0 2JF9 1Y1M 1R5K1UOM 1ERR 2IOK 3UUC 1Y1N 2AYR 3ERT 2BJ4 0.804 2JFA 1.196 0.554 1SJ00.786 0.637 1.115 2JF9 1.177 0.411 0.415 1.186 1Y1M 0.978 0.687 1.1180.276 1.072 1R5K 1.483 0.759 0.52 1.307 0.892 1.342 1UOM 0.739 0.7610.723 0.489 0.909 0.499 1.115 1ERR 1.12 0.483 0.595 1.016 0.851 1.1121.208 0.918 2IOK 0.824 0.689 0.787 0.899 0.897 0.854 1.208 0.736 0.8383UUC 1.024 0.915 0.896 1.03 0.888 1.036 1.228 1.012 0.873 0.929 1Y1N0.749 0.683 1.105 0.432 1.061 0.318 1.293 0.557 1.076 0.744 1.015 2AYR0.659 0.682 0.95 0.792 1.124 0.777 1.391 0.491 1.118 0.071 1.031 0.581

TABLE 12 Analysis of ligand binding in ER-α LBD-antagonist complexesEC₅₀ Ligand: Binding to (μM) Comments 3ERT OHT: E353, R394 0.010 Flippedamine, F404 was too far from the phenol thus there were nopi-interactions 2BJ4 OHT: E353, R394, pi 0.010 F404 2JF9 OHT: E353,D351, 0.010 H524, pi F404 2JFA RAL: E353, D351, 0.002 H524 and pi F404×2 1ERR RAL: E353, D351, 0.002 Phenol flipped for H524 R394 and pi F404×2 1YIM CM3: E353, H524, 0.0015 D351-carboxyle oriented D351 pi F404(IC₅₀) well with pyrrolidine 1YIN CM3: E535, H524 pi 0.001 F404 1SJ0E4D: E353, H524, pi 0.0008 F404 ×2 (IC₅₀) 1R5K GW5: D351 pi F404 0.039No anchor bond with E353 (IC₅₀) 1UOM PTI: E353, H524 pi NA F404 2IOKIOK: E353 pi F404 0.001 3UUC OD1: E353, R394, NA Very small compoundT347 2OUZ C3D: E353, pi F404 0.003 2AYR L4G: E353, pi 0.0107 F404 ×2

TABLE 13 Model evaluation for RAD1901 docking Can model EF₅₀ predictLigand EC₅₀ (=predictive crystal docking RAD1901 (μM) power) structure?score docking score 1ERR 0.001 No −11.452 −7.912 3ERT 0.002 No −12.175−8.151 3UCC NA 8474 Yes −9.278 NA 2IOK 0.001 Yes −11.952 −10.478 1R5K0.039 6100 Yes −11.518 −12.102 1SJ0 0.001 7511 Yes −12.507 −9.816 2JFA0.001 6780 Yes −11.480 −11.055 2BJ4 0.002 5642 Yes −9.727 −11.971 2OUZ0.003 — Yes −11.789 −9.611

TABLE 14 Induced Fit Docking Score of RAD1901 with 1R5K, 1SJ0, 2IFA,2BJ4 and 2OUZ ER-α Crystal Structure RAD1901 IFD Docking Score 1R5K−14.1 1SJ0 −13.1 2JFA −13.9 2BJ4 −13.8 2OUZ −13.4

1-18. (canceled)
 19. A method of treating breast cancer in a subjecthaving a estrogen receptor alpha-positive cancer that is drug-resistantand/or has a mutant estrogen receptor alpha comprising administering tosaid subject a therapeutically effective amount of a combination of anm-TOR inhibitor and RAD1901 having the structure:

or a salt or solvate thereof.
 20. The method of claim 19 wherein saiddrug resistant breast cancer is resistant to one or more antiestrogenand/or or aromatase inhibitor therapies.
 21. The method of claim 20wherein said one or more antiestrogens are selected from the groupconsisting of tamoxifen, toremifene and fulvestrant and said one or morearomatase inhibitors are selected from the group consisting of aromasin,letrozole and anastrozole.
 22. The method according to claim 19 whereinsaid subject expresses at least one mutant estrogen receptor alphaselected from the group consisting of D538G, Y537S, Y537N, Y537C, E380Q,S463P, L536R, L536Q, P535H, V392I and V534E.
 23. The method of claim 22wherein said mutant estrogen receptor alpha is selected from the groupconsisting of Y537S, Y537N, Y537C, D538G, L536R, S463P and E380Q
 24. Themethod according to claim 22 wherein said mutant receptor alpha isY537S.
 25. (canceled)
 26. The method according to claim 19 wherein saidRAD1901 is administered in a total daily dosage of from between 100 mgand 1,000 mg.
 27. The method according to claim 26 wherein said RAD1901is administered in a total daily dosage of 100 mg, 200 mg, 300 mg, 400mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg or 1,000 mg.
 28. The methodaccording to claim 22 wherein said daily dosage is delivered in twoseparate doses.
 29. The method according to claim 28 wherein saidseparate doses are equal doses. 30-31. (canceled)
 32. The methodaccording to claim 19 wherein said woman is post-menopausal.
 33. Themethod according to claim 19 wherein said woman is first identified fortreatment through measuring for increased expression of one or moregenes selected from ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM,AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3,CCNE1, CDH1, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA,CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1,EWSR1, FBXW7, FGF4, FGFR1, FGFR2, FGFR3, FLT3, FRS2, HIF1A, HRAS, IDH1,IDH2, IGF1R, JAK2, KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4,MCL1, MDM2, MDM4, MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2,NKX2-1, NOTCH1, NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD,RARA, RB1, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11,TET2, TP53, TSC1, TSC2, and VHL.
 34. The method according to claim 33wherein said one or more genes is selected from AKT1, AKT2, BRAF, CDK4,CDK6, PIK3CA, PIK3R1 and MTOR.
 35. The method according to claim 19wherein said m-TOR inhibitor is selected from the group consisting ofsirolimus, temsirolimus, everolimus, and ridafarolimus.
 36. The methodaccording to claim 19 wherein said m-TOR inhibitor is dosed at frombetween 1 mg and 500 mg daily.
 37. The method according to claim 36wherein said m-TOR inhibitor is dosed at from between 5 mg and 100 mgdaily.
 38. (canceled)
 39. The method according to claim 35 wherein saidm-TOR inhibitor is everolimus.
 40. The method according to claim 39wherein said everolimus is dosed at a daily dose of 10 mg.
 41. Themethod according to claim 39 wherein said everolimus is dosed at a dailydose from between 2.5 mg and 7.5 mg.
 42. (canceled)
 43. The methodaccording to claim 19 wherein said m-TOR inhibitor is dosed once perday.